Methods of treating a hemolytic disorder comprising administering anti-properdin antibodies

ABSTRACT

A method of treating a hemolytic disorder in a subject in need thereof includes administering to the subject a therapeutically effective amount of an antibody that binds to a component of alternative pathway C3 convertase and selectively inhibits C3a, C5a, C3b, C5b, and C5b-9 produced exclusively by the alternative pathway, without inhibiting any of the classical pathway&#39;s ability to produce C3a, C5a, C3b, C5b, and C5b-9.

RELATED APPLICATION

This application claims priority benefit of U.S. Provisional PatentApplication Ser. No. 61/709,796, filed on Oct. 4, 2012, the content ofwhich is incorporated herein by reference in its entirety.

BACKGROUND

The complement system can be activated through three distinct enzymaticcascades, referred to as the “classical pathway”, “Lectin/MBL”, and“alternative” pathway” (CP, MBL, and AP respectively). MBL is notdiscussed here. The classical pathway is responsible for aiding in hostdefense against antigens to prevent infection of cells. The lectinpathway is a variation of the classical pathway. The alternative pathwayis currently thought to be responsible for 80-95% of total complementactivity in cases where trigger of complement activation is theclassical pathway (“AP amplification loop”). The alternative pathway byitself is activated in a number of disease indications where complementcomponents have been found in elevated state.

There are three “alternative pathway specific proteins”; Factors B, D,and P, which play a major role in the; a) initiation and propagation ofthe alternative pathway and b) classical pathway propagation via thealternative pathway amplification loop. Proteins C3 and C3b, the keyplayers in complement system, are common to all classical andalternative complement pathways. While there may be a one type of C3,there are three different types of C3b produced as each C3 convertase isdifferent. AP C3 convertase is composed of PC3bBb, the classical C3convertase is made up of different proteins. Therefore it is hard tobelieve that the cut would be all identical to produce similar C3bmolecules. As a result, C3b produced by the alternative pathway isdifferent compared to C3b produced via the classical pathway.

The classical pathway (CP) is initiated by antigen-antibody complex. TheCP progression involves proteins such as C1Q, C1r/C1s, C4, and C2. TheCP C3 convertase consists of C3bC4b2a. This complex can cleave the C3into C3b and C3a. This C3b is derived from classical pathway convertaseand is usually required for opsonization of various pathogens andbacteria. Inhibition of this C3b is undesirable. C3b coated cells areremoved via complement receptors present on various cells.

Both complement pathways independently produce C3a, C3b, C5a, C5b,C5b-9, and sC5b-9 as complement activation byproducts.

During classical pathway triggered activation of the alternativepathway, Classical pathway C3 convertase also cleaves C3 into C3b whichcan work independent of the alternative pathway with full amplificationof the classical pathway in 1% normal human serum in the presence ofCa²⁺/Mg²⁺ ions. Classical pathway C5 convertase can cleave C5 togenerate C5a and C5b. The C5b molecule then inserts into the lipidbilayer of the cell to initiate the formation of C5b-9 or sC5b-9.

In alternative pathway activation, C3b produced by the complement systemcan bind properdin and Factor B to form the complex “PC3bB”. Factor Dthen cleaves Factor B, within the complex, into Bb and Ba. This cleavageresults in the release of Ba from the complex and the formation of theAP convertase PC3bBb. PC3bBb cleaves C3 into C3a and C3b, therebyperpetuating the amplification loop of the alternative pathway for thebenefit of the alternative pathway. PC3bBb can then cleave C5 to makeC5b and C5a. The C5b molecule then inserts into a lipid bilayer of acell and forms the nucleus for MAC deposition.

The classical pathway can also initiate the propagation of a part of thealternative pathway known as the amplification loop. Within theamplification loop, C3b binds properdin and Factor B to form the complex“PC3bB”. Factor D then cleaves Factor B, within the complex, into Bb andBa. This cleavage results in the release of Ba from the complex and theformation of the AP convertase PC3bBb. PC3bBb cleaves C3 into C3a andC3b, thereby perpetuating the amplification loop.

C3b is therefore both a component and a byproduct of the complementsystem irrespective of the type of complement pathway activation. Duringthe amplification of the AP, as the PC3bBb (AP C3 Convertase) generatesincreasing amounts of C3b, an amplification loop is established so thatactivation of the alternative pathway can continue. Furthermore, theclassical pathway can also generate C3b, which can bind factor B andthereby engage the alternative pathway, even though the trigger is CPmediated. This allows more C3b to deposit on a target, which leads toenhanced amplification of AP activation.

Addition of newly formed C3b to the existing AP C3 convertase PC3bBbgenerates the AP C5 convertase. Addition of newly formed C3b to theexisting CP C3 convertase generates CP C5 convertase. Both C5convertases have the ability to cleave C5 to produce C5b and C5a. Theterminal complex produced as a result of complement activation is knownas the MAC complex (also known as C5b-9 or sC5b-9), which is responsiblefor lysis of cells in a subject. Both C3a and C5a are potentanaphylatoxins that are responsible for activating platelets,neutrophils, and monocytes. As a result, inflammatory molecules such aselastase, TNF-α, IL-1, VEGF, and peroxides are released. Formation ofC5b-9/sC5b-9 is responsible for tissue damage and tissue injury/tissuedamage seen in “other diseases”

Classical complement pathway activation provides a valuable first-linedefense against potential pathogens and can generate C3a/C3b, C5a/C5b,and C5b-9/sC5b-9. Therefore, exacerbation of the classical pathway canproduce large amounts of complement byproducts. As described elsewhere,both C3a and C5a are potent anaphylatoxins, C3b mediates opsonization,and C5b is responsible for wanted killing of the pathogens. Here, bothC3a and C5a would generate beneficial responses and are produced to killthe invaders. This pathway is required for host defense and thereforemust not be inhibited.

Alternative pathway activation in Mg++ ions, without the calcium ions,guarantees only the AP activation. In disease state, this pathway isactivated independent of the classical pathway. This pathway is notrequired for host defense and therefore can be inhibited in itsentirety.

SUMMARY

Embodiments described herein relate to antibodies that prevent C3bformation responsible for extravascular hemolysis and C5b-9 responsiblefor intravascular hemolysis. The invention further relates to methodsfor treatment of subjects suffering from disorders that involve lysis ofred blood cells and platelets via intravascular and extravascular route.The invention also covers protection of neutrophils, monocytes,platelets, and T-lymphocytes against complement attack. This isaccomplished by antibodies of the claimed genus that block the formationand deposition of C3b on cells and C5b-9 on cells that are deficient inGPI linked proteins.

This application summarizes a group of complement inhibitor monoclonalantibodies that prevent the formation of alternative pathway derived C3band C5b-9 formation. These antibodies are being claimed as a genus inthis particular application. Although these antibodies bind differenttargets within the alternative pathway, they have unique feature as theyall inhibit alternative pathway generated C3b called ‘C3b” but not theclassical pathway generated C3b.

It is Removal of cells causes cytopenia depending upon the cell typeunder attack—neutropenia, monocytopenia, thrombocytopenia,lymphocytopenia, and leukopenia. Thus, inhibition of AP activation by aclaimed genus of monoclonal antibodies can prevent cytopenia in asubject (human). Cytopenia is commonly observed in hematologicaldisorders such as Paroxysmal Nocturnal Hemoglobinuria (PNH), IdiopathicThrombocytopenic Purpura (ITP), Thrombotic Thrombocytopenic Purpura(TTP), Hemolytic-Uremic Syndrome (HUS), Disseminated IntravascularCoagulation (DIC), Antiphospholipid Syndrome (APS), Post-TransfusionPurpura, Neonatal Allo-Immune Thrombocytopenia (NAITP). The antibodiesof the claimed genus are capable of preventing cytopenia, cellularactivation, cell dysfunction, inflammation, extravascular hemolysis,intravascular hemolysis and tissue injury.

Both the classical and the alternative pathways upon activation produceC3b molecules. The two C3b although called the same but are different.C3b molecules produced by alternative pathway but not the classicalpathway in PNH bind erythrocytes, neutrophils, monocytes, platelets, andT lymphocytes. This binding results in clearance of such cells viaextravascular hemolysis. Removal via extravascular hemolysis causescytopenia and increased levels of bilirubin and LDH. C5b-9 (also knownas MAC) deposits onto the cell membrane and results in lysis ofanucleated cells such as erythrocytes and platelets. Clear evidence ofC3b deposition and C5b-9 deposition have not been reported previously.Erythrocyte lysis results in increased LDH levels, increasedreticulocyte counts and decreased levels of hemoglobin in erythrocytes.

In PNH, we found that C3b and C5b-9 have been associated with bothanucleated and nucleated cells. These patterns of C3b and C5b-9 bindingto a variety of cells deficient in GPI linked proteins suggestsdestruction/partial destruction, activation, or dysfunction of suchcells. Current invention is to prevent the formation and deposition ofsuch molecules on a variety of anucleated and nucleated cells that areresponsible for pathological outcomes in diseases where the absence ofGPI liked proteins is associated with pathology. Neutralizing antibodiesthat prevent the formation of C3b and C5b-9 via the alternative pathwayare covered under this invention.

As an example of hematological disorder where cytopenia occurs is PNH.Cytopenia covers leukopenia, neutropenia, monocytopenia,thrombocytopenia, lymphocytopenia. Nearly all types of cells appear tobe deficient in GPI linked proteins in PNH. Such cells are subject tocomplement attack via C3b deposition and extravascular removal and/ordestruction via extravascular route. All antibodies that are selectiveblocker of only alternative pathway-derived C3b and C5b-9 are coveredunder this invention. A set of such antibodies that perform suchfunction are covered under this invention.

T-lymphocytes, monocytes, and neutrophils are all deficient in GPIlinked proteins and therefore are subject to complement attack anddeposition of C3b and C5b-9. C3b coated cells would be deprived of theproper function and C5b-9 deposition would cause cell death resulting inloss of the cells. As an example, the neutrophil is the key cellfighting bacterial and fungal infection in the body. These neutrophilsin PNH patients may not ingest germs effectively and are therefore lessable to fight infection. These patients, whose white blood cells don'twork properly, are much more likely to develop a second infection. It isknown via the laboratory testing that by adding GM-CSF in the laboratorytesting, it is possible to restore the ability of the white blood cellsto ingest bacteria and fight infection. Thus addition of GM-CSF isproposed as a potential use in patients to increase the ability ofneutrophils to behave normally. The main function of GM-CSF is known.The protein is a cytokine that functions as a white blood cell growthfactor in general. GM-CSF stimulates stem cells to produce granulocytes(neutrophils, eosinophils, and basophils) and monocytes. Thus increasedamount of GM-CSF seen in the PNH blood samples indicates that PNH cellsare dying. Thus adding GM-CSF with and without the claimed genus ofantibodies could help improve the cell quality in general and increasethe ability of cells to fight infections.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic showing the complement cascade of the classicalpathway and the alternative pathway. Lectin pathway is not shown as itis not the part of the invention. The FIG. 1 shows a schematicrepresentation of the CP and the AP. In this figure, we show that bothCP and AP are distinct and not connected. The schematic only shows howthe antibodies of the current genus work and not the way antibodies ofthe other invention work. AP amplification is shown in the upper righthand side and consists of PC3b, PC3bB, PC3bBb. As can be seen in theschematic, PC3bBb then acts to perpetuate the cycle by cleaving C3 intomore C3b which binds to P to again form PC3b. Application of theinvention completely inhibits the alternative pathway without affectingthe classical pathway by specifically targeting the components of thisamplification loop. These antibodies prevent the amplification loop ofthe alternative complement pathway without affecting the classicalpathway (as shown on the left side of the schematic in FIG. 1).

Based on the old convergence theory describing C3 being the convergencepoint, those with ordinary skill in the art would expect any activationof the classical pathway to invariably have the effect of alternativepathway activation. This is because the two pathways are believed to“overlap” at the starting point of the C3. According this theory, C3bproduced via the classical pathway participates in the AP amplificationloop. The invention that is the subject of this patent is thedevelopment of a new and unique genus of complement inhibitingantibodies which challenge that assumption. The claimed invention, thisnew genus of antibodies, specific targets components of the alternativepathway amplification loop in such a way as to inhibit the alternativepathway regardless of whether or not the AP amplification loop has beenotherwise triggered by the classical pathway. Thus anti-C3b antibodiesof the current invention only inhibit the AP and not the CPamplification loop or the CP propagation. The uniqueness of theinvention is not only which components these antibodies target, but howthey target those components. Similarly, we describe the anti-Ba,anti-Bb, and -P and anti-C3b for the invention.

FIG. 2 illustrates three assay figure tracings from real data generatedfrom one of the invented antibodies as a representative FIG. 4. One linerepresents untreated sample whereas the second line represents theantibody treated sample. The Panel A is a CP assay conducted in 1% NHSin CP buffer. The second panel is a CP assay in 10% NHS that allows CPamplification loop to contribute into the AP. The third panel (Panel C)shows inhibition by the invented antibodies of the genus that inhibitthe AP without affecting the CP (Panel B). All antibodies showing thispattern would belong to the invented genus.

FIGS. 3 (a-e) are plots showing the binding affinities of the inventedantibodies to their respective targets (C3b, Bb, and P).

FIG. 4 is a graph showing that the invented antibodies inhibitalternative pathway dependent hemolysis of rabbit erythrocytes (rRBC) inHuman Serum (NHS). There exist a multitude of antibodies which inhibitthe activities of Properdin (Factor P), Factor Bb, and C3b. All suchantibodies inhibit the alternative pathway and not the CP (FIG. 7).However, these antibodies will act on their targets in such a way as toinhibit the alternative pathway without inhibiting the classicalpathway.

FIG. 5 is a graph showing that the invented antibodies do not inhibitclassical pathway dependent lysis of Antibody Sensitized SheepErythrocytes (sRBC). The current state of the act teaches thatactivation of the classical pathway invariably results in activation ofthe alternative pathway at the amplification loop, which begins withcleavage of C3 by CP produced C3 convertase. The claimed invention makespossible the therapeutic inhibition of the alternative pathway, despiteclassical pathway activity. As shown, the Anti-C3b, Anti-Ba, Anti-Bb,and Anti-P antibodies of the invented genus do not inhibit the classicalpathway and are specific to the alternative complement pathway (FIGS.2-3). Therefore, the invention could have potential application in anydisease characterized or mediated by a pathological over-activation ofthe alternative complement pathway.

FIGS. 6 (a-c) illustrate plots showing that the invented antibodiesinhibit the formation of C3b in serum, a marker for extravascularhemolysis.

FIGS. 7 (a-c) illustrate plots showing that the invented antibodiesinhibit the formation of C5b-9 in serum, a marker for intravascularhemolysis.

FIG. 8 is a graph showing that the invented antibodies inhibit theformation of C3a in Whole Blood Inflammation. Both C3a (cleaved from C3)and C5a (cleaved from C5) are potent anaphylatoxins (triggers of localinflammation) that are produced upon complement activation. Both theclassical pathway and the alternative pathway produce these molecules.The Figure shows the inhibition of C3a derived from the alternativecomplement pathway. Classical pathway trigger does not exist in thismodel.

FIG. 9 is a graph showing that the invented antibodies inhibit theformation of C5a in Whole Blood Inflammation. The claimed inventionselectively inhibits C3a (FIG. 8) and C5a (FIG. 9) produced from thealternative pathway.

FIG. 10 is a graph showing that the invented antibodies Inhibitformation of sC5b-9 in Whole Blood.

FIG. 11 is a graph showing that the invented antibodies inhibitneutrophil activation. The neutrophils activation occurs due to theactivation of the AP and not CP or CP-induced AP.

FIG. 12 is a graph showing that the invented antibodies inhibit monocyteactivation. The monocyte activation occurs due to the activation of theAP and not CP or CP-induced AP.

FIG. 13 is a graph showing that the invented antibodies inhibit plateletactivation. The platelet activation occurs due to the activation of theAP and not CP or CP-induced AP.

FIG. 14 is a graph showing that the invented antibodies inhibitmonocyte-platelet aggregates. The monocyte-platelet aggregation occursdue to the activation of the AP and not CP or CP-induced AP.

FIG. 15 is a graph showing that the invented antibodies inhibit elastaserelease from neutrophils. The neutrophil elastase is produced fromneutrophils that are activated via the C3a/C5a produced from thealternative pathway.

FIG. 16 is a graph showing antibodies of the invention prevent LDHrelease in an in vivo model of PNH.

FIG. 17 is a graph showing antibodies of the invention prevent HgBrelease in vivo in a model of PNH.

FIG. 18 illustrates a graph showing cells in blood from PNH patients.

DETAILED DESCRIPTION Definitions

Unless specifically defined herein, all terms used in this document havethe same meaning as would be understood by those of ordinary skill inthe art of the present invention. The following definitions are providedfor clarity, and to define their intended meaning as used in thespecification and claims to describe the present invention.

Complement Pathways

“CLASSICAL PATHWAY” refers to complement which is triggered byantigen-antibody complexes for activation and may or may not alsotrigger the alternative pathway amplification loop for its propagation.

“ALTERNATIVE PATHWAY” refers to complement activation which is triggeredby a cell surface (or cell-surface like material) looking like a foreignsurface. The absence of GPI linked protein makes the surface of the PNHcell foreign enough to activate the alternative pathway. The alternativepathway may also begin with spontaneous proteolytic generation of C3bfrom complement factor C3, where C3b has the ability to bind B and Pboth.

“ALTERNATIVE PATHWAY SPECIFIC PROTEIN” refers to C3b, factor B, factorBb, factor D, and/or properdin. Here C3b refers to C3b as a part of theAP and not CP.

“AP AMPLIFICATION LOOP” refers to a looping series of reactions in whichC3b formed makes AP C3 convertase. This convertase cleaves C3 andgenerates more C3b, which feeds back into the loop. Thisself-perpetuating cycle of reactions generates large amounts of C3b.

“C3b” is term used for both C3b derived from AP and CP pathways.

“ALTERNATIVE PATHWAY-DEPENDENT C5a” describes the formation of C5aproduced from activity of the alternative pathway of the complementsystem in whole blood. For example, “AP-dependent C5a formation” refersto the formation of C5a via activation of the alternative pathway, whichis independent of the classical pathway.

“ALTERNATIVE PATHWAY-DEPENDENT sC5b-9” describes the formation of sC5b-9produced from activity of the alternative pathway of the complementsystem. For example, “AP-dependent sC5b-9 (soluble MAC) formation”refers to the formation of sC5b-9 via activation of the alternativepathway, which is independent of the classical pathway.

“ALTERNATIVE PATHWAY-DEPENDENT C5b-9” describes the formation of C5b-9produced from activity of the alternative pathway of the complementsystem. For example, “AP-dependent C5b-9 formation (Deposited MAC)”refers to the formation of C5b-9 via activation of the alternativepathway, which is independent of the classical pathway.

“C3a DEPENDENT CELLULAR ACTIVATION” describes the activation ofneutrophils, monocytes, platelets, T lymphocytes, endothelial cells,mast cells, and platelets which occurs when AlternativePathway-Dependent C3a binds to C3a receptors, which are present on thesecells. These cells are found, in their C3a activated state, in variousdifferent diseases (see OTHER DISEASES).

“C5a DEPENDENT CELLULAR ACTIVATION” describes the activation ofneutrophils, monocytes, platelets, T lymphocytes, endothelial cells,mast cells, and platelets which occurs when AlternativePathway-Dependent C5a binds to C5a receptors, which are present on thesecells. These cells are found, in their C5a activated state, in variousdifferent diseases (see “OTHER DISEASES”).

“C5b-9 and sC5b-9 DEPENDENT TISSUE INJURY/CELLULAR DAMAGE” describes thecellular damage caused by the formation of sC5b-9 and/or C5b-9. Thesemolecules either bind to the cellular surface and/or insert themselvesinto the cell's plasma membrane resulting in pathological conditionsalso described as “TISSUE INJURY”. Tissue injury occurs in variousdiseases and can result in the damage to various organs.

“MEMBRANE ATTACK COMPLEX” (“MAC”) refers to a complex of the terminalfive complement components (C5b-C9) that inserts into and disrupts cellmembranes. This complex is also referred to as C5b-9. MAC complex isproduced by both the alternative pathway and by the classical complementpathway. The complex that is associated with “S protein” is calledsC5b-9, a soluble form of MAC. The invented antibodies inhibitalternative pathway associated C5b-9 and sC5b-9.

“C3a, C5a, C5b-9, sC5b-9 AND INFLAMMATION” describes inflammation causedby the products of AP activation and activity; and in particular, the APproducts C3a, C5a, C5b-9, and sC5b-9 generating from AP activity. Thesemolecules cause C3a DEPENDENT CELLULAR ACTIVATION, C5a DEPENDENTCELLULAR ACTIVATION, C5b-9 and sC5b-9 DEPENDENT CELLULAR DAMAGE, andresult in the prevalence of CYTOKINE ACTIVATED CELLS, PROTEASE ACTIVATEDCELLS, and PEROXIDE ACTIVATED CELLS, all of which can be implemented invarious different diseases and disease pathologies.

Whole Blood & Inflammation

“WHOLE BLOOD” describes complete blood with the same composition ofcells, chemicals, proteins, etc. as blood found in human blood vessels.The isolated blood contains all components of the complement systemincluding inflammatory cells that are responsible for inflammatoryresponses.

“INFLAMMATION IN WHOLE BLOOD” describes the cascade of reactionsbeginning with alternative pathway activation in whole blood, theresulting production of C3a, C5a, and C5b-9 and sC5b-9 in whole blood,the resulting activation of neutrophils monocytes and platelets in wholeblood, and ultimately, the production of inflammatory cytokines in wholeblood (in vivo or ex vivo). Several inflammatory mediators are found tobe secreted into the plasma. These inflammatory mediators are TNF, IL-1,IL-6, IL-8 and several others. Not included in the list.

“ALTERNATIVE PATHWAY (AP)-DEPENDENT INFLAMMATION IN HEMOLYTIC DISEASES”refers to an increase in alternative complement pathway activity, asmeasured by continued or increased formation, and/or release, of one ormore of the following components C3a, C3b, C5a, C5b-9, or sC5b-9, andall the anticipated consequences thereof, in PNH and other hemolyticdiseases. Such anticipated consequences include; continued or increasedAP-dependent MAC-mediated deposition and/or lysis of cells, continued orincreased AP-dependent activation of platelets, monocytes, neutrophils,mast cells, or basophils; and/or continued or increased AP-dependentformation or release of TNF-α, IL-1, or neutrophil elastase.

“OTHER ORPHAN AND NON_ORPHAN HEMATOLOGICAL AND NON HEMATOLOGICAL ACUTEAND CHRONIC DISEASES” describes a list of diseases where one of theelevated components measured is derived from the activation of thealternative pathway system. These components include but not limited to;C3a/C3b, P, Ba/Bb, C5a/C5a, and C5b-9/sC5b-9. Elevated levels of thesecomponents have been found associated with one or more diseases. Thesecomponents are responsible for cellular activation and release ofinflammatory mediators. These, in turn, ultimately cause tissue damage,defining the disease in both hematological and non-hematologicaldiseases.

“ALTERNATIVE PATHWAY (AP)-DEPENDENT INFLAMMATION IN PNH” refers to anincrease in alternative complement pathway activity, as measured bycontinued or increased formation, and/or release, of C3a, C3b, C5a, C5b,C5b-9, and/or sC5b-9, and all the anticipated consequences thereof. Suchanticipated consequences include; continued or increased AP-dependentC3b and MAC-mediated deposition or lysis of cells, continued orincreased AP-dependent activation of platelets, monocytes, neutrophils,mast cells, or basophils; and/or continued or increased AP-dependentformation or release of TNF-α, IL-1, or neutrophil elastase.

“AUTOIMMUNE DISEASE” refers to a condition where the immune response ofa subject is inappropriately directed against substances and tissuesnormally present in the body.

“CELLULAR LYSIS” indicates tissue injury in part. Cellular lysis occursas a result of C5b-9 formation of the cell surface. Such deposition ofC5b-9 leads to cellular injury and in case of tissues the cell injury isa tissue injury.

Inhibitory Antibodies and Agents

“AGENT” “COMPOUND” refers to any substance, molecule, element, compound,entity, or any combination thereof. An agent can be, among other things,a protein, oligopeptide, small organic molecule, polysaccharide,polynucleotide, or other biochemical substance. It can be a naturalproduct, a synthetic compound, a chemical compound, or a combination oftwo or more substances of different origins. Unless otherwise specified,the terms “agent”, “substance”, and “compound” can be usedinterchangeably.

“Alternative pathway specific antibody” refers to an antibody orfragment thereof that can bind to an alternative pathway protein toinhibit activation and/or progression of the alternative pathway in asubject.

“ANTIBODIES TO AP PROTEINS” describe anti-P, anti-Ba, anti-Bb, anti-C3bantibodies that neutralize the activity of the alternative pathwaywithout inhibiting the classical pathway.

Pharmacology

“PHARMACOKINETIC ACTIVITY” or “PHARMACOKINETICS” refers to themechanisms of absorption and distribution of an administered drug, therate at which a drug action begins and the duration of the effect, thechemical changes of the substance in the body, and the effects androutes of excretion of the metabolites of the drug

“THERAPEUTICALLY EFFECTIVE AMOUNT” is defined as an amount sufficient tocompletely inhibit AP activity in vivo

As used herein, a “prophylactically effective amount” is defined as anamount sufficient to prevent the onset of a disease or disorder in asubject.

As used herein, the terms “administering,” “administration,” and likerefer to ways in which the antibody or antigen binding fragment thereofcan be given to the subject, including, but not limited to, oraladministration, intravenous administration, intraperitoneal,intramuscular, subcutaneous administration, aural administration, orrectal administration.

Antibodies

“ANTIBODY” used in the broadest sense includes monoclonal antibodies,including full length or partial length monoclonal antibodies, andpolyclonal antibodies from mouse, rabbit or human species. Theantibodies can also be egenrated in other mammalas. In its most widelyrecognized form, an antibody contains two heavy (H) chains and two light(L) chains inter-connected by disulfide bonds. Each heavy chain iscomprised of a heavy chain variable region (abbreviated herein as VH)and a heavy chain constant region. The heavy chain constant region iscomprised of three domains, CH1, CH2 and CH3. Each light chain iscomprised of a light chain variable region (abbreviated herein as VL)and a light chain constant region. The light chain constant region iscomprised of one domain, CL. The VH and VL regions can be furthersubdivided into regions of hyper-variability, termed complementaritydetermining regions (CDRs), interspersed with regions that are moreconserved, termed framework regions (FR). Each VH and VL is composed ofthree CDRs and four Frameworks arranged from amino-terminus tocarboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4. The variable regions of the heavy and light chains contain abinding domain that interacts with an antigen. The term “antibody”encompasses whole antibodies and antibody fragments thereof, derivedfrom any antibody-producing mammal (e.g., mouse, rat, rabbit, andprimate including human), that specifically bind to proteins such asproperdin, C3b, Ba, and Bb or portions thereof. Exemplary antibodiesinclude polyclonal, monoclonal and recombinant antibodies;multi-specific antibodies (e.g., bispecific antibodies); humanizedantibodies; murine antibodies; chimeric, mouse-human, mouse-primate,primate-human monoclonal antibodies; and anti-idiotype antibodies, andmay be any intact molecule or fragment thereof.

“OTHER ANTIBODIES” refer to antibodies developed in living organismincluding and not limited to animals and humans for therapeutic use inhumans and animals. Any antibodies raised in a living organism iscapable of inhibiting AP mediated lysis (Assay-3) but not the CPmediated lysis or the CP amplification loop.

“ANTIBODY FRAGMENT” refers to a portion derived from or related to afull-length antibody, particularly an anti-C3b, anti-P, and anti-Ba, oranti-Bb antibody, generally including the antigen binding or variableregion thereof (see “ANTIGEN BINDING FRAGMENT”). The term “antibodyfragment” refers to a portion derived from a full-length alternativepathway inhibitory antibody, generally including the antigen binding andvariable region thereof. Other antibodies include nano bodies,diabodies, linear antibodies, single-chain antibody molecules andmultispecific antibodies formed from antibody fragments. Examples ofantibody fragments include Fab, Fab′, F(ab)2, F(ab′)2 and Fv fragments,or scFv fragments (and any PEGylated variations of any of the forgoing).

“ANTIGEN BINDING FRAGMENT” of an antibody refers to the one or morefragments of an intact antibody that retain the ability to specificallybind to a given antigen. Antigen binding functions of an antibody can beperformed by fragments of an intact antibody containing theComplementarity Determining Regions (CDRs). Examples of antigen bindingfragments:

-   -   “Fab” fragments (single chain variable regions with VH and VL);    -   “Monovalent Fragments” (antibody fragments consisting of the VL,        VH, CL and CH1 domains);    -   “F(ab′)2” fragments (bivalent fragments comprising two Fab        fragments linked by a disulfide bridge at the hinge region);    -   “Fd” fragments (which consist of the VH and CH1 domains of an        antibody);    -   “Fv” fragment (which consist of the VL and VH domains of a        single arm of an antibody);    -   single domain antibody (“dAb”), which consist of a VH domain or        a VL domain;    -   an isolated Complementarity Determining Region (“CDR”).

A “FUNCTIONAL DERIVATIVE” of an antibody is any compound which is eithertaken from, or incorporates within itself, the functional region of theantibody. Functional derivatives of antibodies include, but are notlimited to, antigen binding fragments, CDRs, humanized antibodies, “Fab”fragments, “Fd” fragments, chimeric antibodies, monoclonal antibodies,recombinant antibodies, and single chain antibodies.

CDRs, as antigen binding fragments, can also be incorporated into singledomain antibodies, maxi bodies, mini bodies, intrabodies, diabodies,triabodies, tetra bodies, v-NAR and bis-scFv. Antigen binding fragmentsof antibodies can be grafted into scaffolds based on polypeptides suchas Fibronectin type III (Fn3). Antigen binding fragments can beincorporated into single chain molecules comprising a pair of tandem Fvsegments (VH-CH1-VH-CH1) which, together with complementary light chainpolypeptides, form a pair of antigen binding regions.

As used herein, the term “Fc region” refers to the region of theantibody that induces effector functions.

“AFFINITY” refers to the chemical strength of the interaction between anantibody and an antigen at single antigenic sites.

“BINDING SPECIFICITY” refers to the ability of an individual antibody orantigen binding fragment to bind to a particular target, e.g., thebinding specificity of an antibody to bind only to its target.

“COMPOUNDS,” “BLOCKER”, “INHIBITOR”, or “ANTAGONIST” refers to achemical substance, or force, that retards or prevents a chemical orphysiological reaction or response. Common blockers or inhibitorsinclude, but are not limited to, antisense molecules, antibodies,antagonists and their derivatives. For example, an antibody that bindsto a component of an AP specific interaction between that component andanother component of the AP. Such an antibody would be an inhibitor orblocker of that interaction and, by extension, the AP.

“CHIMERIC ANTIBODY” is a recombinant protein that contains the variabledomains and CDRs derived from an antibody of from a non-human species ofanimal, while the remainder of the antibody molecule is derived from ahuman antibody. The replacement of the non-binding region of theantibody with a human constant region enables the chimeric antibody toretain its specificity in recognizing and binding the targeted antigenwhile having reduced antigenicity in humans (compared to the originalmouse antibody).

“HUMANIZED ANTIBODY” is an antibody that consists of non-human CDRs andhumanized framework regions. Humanized antibodies are typicallyrecombinant proteins in which only the antibodycomplementarity-determining regions are of non-human origin.

As used herein, a “single-chain Fv” or “scFv” antibody fragmentcomprises the VH and VL domains of an antibody, wherein these domainsare present in a single polypeptide chain.

As used herein, the term immunogenicity refers to the ability of anantigen to initiate an immune response in a subject.

“COMPLEMENTARITY DETERMINING REGIONS (CDRs)” are the key binding regionsof the antibody. There are typically three CDRs found within thevariable regions of each of the two heavy and light chain variableregions. CDRs can be shuffled around, in terms of location, to create aparticular binding affinity. See also “ANTIGEN BINDING FRAGMENTS.”

“EFFECTOR FUNCTIONS” refer to those biological activities attributableto the native Fc region of an antibody, and vary with the antibodyisotype. Examples of antibody effector functions include: C1q bindingand complement dependent cytotoxicity; Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor); lack ofactivation of platelets that express Fc receptor; and B cell activation.In order to minimize or eliminate side effects of a therapeuticantibody, it may be preferable to minimize or eliminate effectorfunctions.

As used herein, the term “reduced Fc effector function(s)” refers to thefunction(s) of an antibody wherein the antibody does not act against anantigen that recognizes the Fc region of the antibody. Examples ofreduced Fc effector functions can include, but are not limited to,reduced Fc binding to the antigen, lack of Fc activation against anantigen, an Fc region that contains mutations to prevent normal Fceffector functions, or prevention of the activation of platelets andother cells that have Fc receptors.

“HUMAN ANTIBODY” is an antibody in which all components of the antibodyare of human origin, including the framework, CDRs, and constantregions. The term “humanized” antibody is an antibody of non-humanorigin that retains the binding specificity of the non-human antibodywhile being less immunogenic in humans. See CHIMERIC ANTIBODY andHUMANIZED ANTIBODY.

“PURIFIED ANTIBODY” refers to antibodies which have been isolated fromcontaminants. In preferred embodiments, the antibody will be purified(1) to greater than 95% by weight of antibody as determined by the Lowrymethod, and most preferably more than 99% by weight, (2) to a degreesufficient to obtain at least 15 residues of N-terminal or internalamino acid sequence by use of a spinning cup sequenator, or (3) tohomogeneity by SDS-PAGE under reducing or non-reducing conditions usingCoomassie blue, or preferably, silver stain.

“ISOTYPE” refers to the antibody class (e.g., IgM, IgE, IgG such as IgG1or IgG4) that is provided by the heavy chain constant region genes.Isotype also includes modified versions of one of these classes, wheremodifications have been made to alter the Fc function, for example, toenhance or reduce effector functions or binding to Fc receptors.

“MONOCLONAL ANTIBODY” refers to an antibody obtained from a populationof substantially homogeneous antibodies, i.e., the individual antibodiescomprising the population are identical. A monoclonal antibody isdirected against a single determinant on the antigen. For example, themonoclonal antibodies useful in the present invention may be prepared bythe hybridoma methodology or they may be made using recombinant DNAmethods in bacterial or eukaryotic animal or plant cells. The“monoclonal antibodies” may also be isolated from phage antibodylibraries, or generated using in vitro, in vivo, and cell culturemethods. Monoclonal antibodies include those that bind to a uniquesequence of amino acids and have a single specific epitope on its targetantigen.

“POLYCLONAL ANTIBODY PREPARATIONS,” unlike monoclonal antibodypreparations, include different antibodies directed against differentdeterminants (epitopes). As used herein, the term “polyclonal” refers toan antibody that recognizes multiple epitope sites on a single antigen.

“RECOMBINANT ANTIBODY” includes all antibodies that are prepared,expressed, created or isolated by recombinant means and methods.

“SINGLE CHAIN ANTIBODY” refers to an antibody in which the two domainsof the Fv fragment, VL and VH, are coded for by separate genes. Thesegenes can be joined, using recombinant methods, by an artificial peptidelinker. Joining the genes results in the production of a single proteinchain in which the VL and VH regions pair to form monovalent molecules(known as single chain Fv, “scFv”). Such single chain antibodies includeone or more “antigen binding fragments” of an antibody. See ANTIGENBINDING FRAGMENT.

“THERAPEUTIC ANTIBODY” refers to an antibody that may be consideredeffective in a therapeutic or prophylactic context with regard to adisease or condition of interest.

Amino Acids and Amino Acid Sequence

“AMINO ACID,” in the broadest sense, refers to the naturally occurringamino acids which can be divided into groups based upon the chemicalcharacteristic of the side chain of the respective amino acids.“Hydrophobic” amino acids are Ile, Leu, Met, Phe, Trp, Tyr, Val, Ala,Cys and Pro. “Hydrophilic” amino acids are, Asn, Gln, Ser, Thr, Asp,Glu, Lys, Arg and His. The “uncharged hydrophilic” amino acids are Ser,Thr, Asn and Gln. The “acidic” amino acids are Glu and Asp. The “basic”amino acids are Lys, Arg and His. As used herein, the amino acidresidues are abbreviated as follows: alanine (Ala; A), asparagine (Asn;N), aspartic acid (Asp; D), arginine (Arg; R), cysteine (Cys; C),glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G), histidine(His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K),methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine(Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y),and valine (Val; V).

“CONSERVATIVE AMINO ACID SUBSTITUTION” is illustrated by a substitutionamong amino acids within each of the following groups: (1) glycine,alanine, valine, leucine, and isoleucine, (2) phenylalanine, tyrosine,and tryptophan, (3) serine and threonine, (4) aspartate and glutamate,(5) glutamine and asparagine, and (6) lysine, arginine and histidine.

“IDENTICAL,” in the context of two or more nucleic acids or polypeptidesequences, refer to two or more sequences or subsequences that are thesame. Two sequences are “substantially identical” if two sequences havea specified percentage of amino acid residues or nucleotides that arethe same (i.e., 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90%,95%, or 99% identity over a specified region, or, when not specified,over the entire sequence), when compared and aligned for maximumcorrespondence over a comparison window, or designated region asmeasured using one of the following sequence comparison algorithms or bymanual alignment and visual inspection. Optionally, the identity existsover a region that is at least about 50 nucleotides (or 10 amino acids)in length, or more preferably over a region that is 100 to 500 or 1000or more nucleotides (or 20, 50, 200 or more amino acids) in length. Thepercent identity between two amino acid sequences can also be determinedusing the algorithm of Meyers and Miller.

PNH and Hemolytic Diseases

As used herein, the term “HEMOLYTIC DISEASES” refers to any disorder ordisease in which cellular lysis, cellular damage and inflammation play arole in the pathology of the disease. Hemolytic disease is also aninflammatory disorder or disease wherein AP activation causes cellularlysis, cellular damage, and inflammation. Hemolytic diseases includediseases characterized by pathologic lysis of erythrocytes and/orplatelets. Anucleated cells such as erythrocytes and platelets aresubject to full lysis. Lysis of erythrocytes releases hemoglobin whichhas pathological outcome for blood and organs. Nucleated cells such asneutrophils, monocytes, T lymphocytes can be attacked by the MAC but donot undergo full lysis.

“INTRAVASCULAR HEMOLYSIS” refers to the lysis of anucleated andnucleated cells which is caused by AP activation and the associatedproduction and deposition of C5b-9 on cell surfaces.

“EXTRAVASCULAR HEMOLYSIS” refers to lysis of cells due to C3b depositionand removal via complement receptors. C3b is produced via the activationof the classical and the alternative pathway. This invention is focusedon C3b produced via the alternative complement pathway.

“TRAP ANTAGONIST” is a receptor-Fc fusion protein consisting of theantibody Fab fused to the Fc portion of human IgG1. In a preferredembodiment, an expression plasmid encoding the target protein istransfected into CHO cells, which secrete the trap antagonist into theculture medium. The resulting antagonist trap binds its ligands usingthe binding domains of high-affinity receptors, having greater affinityfor properdin.

“SUBCUTANEOUS ADMINISTRATION” refers to introduction of a drug under theskin of an animal or human patient, preferable within a pocket betweenthe skin and underlying tissue, by relatively slow, sustained deliveryfrom a drug receptacle. The pocket may be created by pinching or drawingthe skin up and away from underlying tissue. There are variousformulations available specially those skilled in the art are well awareof such formulations.

“TISSUE INJURY” refers to the tissue where C5b-9 (MAC) is found toinjure the tissue. Tissue injury is caused by the MAC and can beinhibited by the antibodies that prevent MAC formation. One exampleshown in the application is the quantifiable death of erythrocytes in atime dependent manner in the presence of normal human serum thatcontains physiological levels of complement components. Thisdemonstration of lysis of cells is quantifiable by the loss ofscattering at OD700. Nucleated cells present in tissues are also injuredby complement similar to erythrocytes. Inhibition of erythrocyte lysisand therefore tissue injury can be prevented by the use of antibodies ofthis invention. Tissue injury can occur in any part of the body/organsand can lead to pathological outcome such as arthritis. In hematologicaldisorder where all cells that lack the GPI are subject to MAC attack,tissue injury and damage can be prevented by the use of such antibodies.This definition can be extended to many diseases where tissue injuryoccurs as a result of AP activation but not CP activation.

Embodiments described herein relate to methods for treating a subjectsuffering from hemolytic disease, hemolytic related, or PNH-like,condition by administering to an afflicted subject an effective amountof one (or several) of a specific genus of inhibitory antibodies thatinhibit intravascular and extravascular lysis mediated only thealternative complement pathway without affecting the classicalcomplement pathway. The antibodies of this genus have been identifiedand selected, from a variety of antibodies inhibiting the complementsystem, for their specific and unique effect on specific components ofthe alternative pathway. The inhibitory antibodies of the claimed genusare selective for the alternative complement pathway. The antibodiesproduced from this combination of selection criteria are useful for amultitude of hemolytic conditions.

Both, the classical and the alternative pathways are independent. Lectinor the MBL pathway is part of the classical pathway. Both pathwaysindependently generate C3b, C3a, C5b, C5a, and C5b-9. Antibodies of thepresent invention inhibit C3b and C5b-9 formation, molecules producedvia both pathways. These monoclonals do not inhibit classical pathwayderived C3b and C5b-9 whether the amplification loop is a part of theprocess or not. This invention leaves the C3b produced via the classicalpathway intact for host defense such as opsonization. This inventionleaves the C5b-9 produced via the classical pathway intact for hostdefense.

Prior art uses inhibitors do not appear to be selective because, theclassical pathway feeds into the alternative pathway and also work inco-ordinance with the alternative pathway. Classical pathway uses theamplification loop of the alternative pathway. Inhibitors of APdeveloped in such a setting would inhibit the amplified activity of theclassical pathway.

Uniquely, complement attack does not damage normal cells, abnormal cellsare those that lack the important regulators of the complement systemsuch as CD55 and CD59. These abnormal cells are found in PNH. PNH cellslack CD55 and CD59, our invention shows that both CD55 and CD59 areabsent in nearly all types of cells including erythrocytes, platelets,T-lymphocytes, neutrophils, and monocytes—but the total population ofeach type of cells may be different—for example, the % of abnormal cellscan vary from less than 1% to 10% or 10% to 100%.

In PNH, abnormal erythrocytes undergo lysis and release hemoglobin as aresult of AP activation. The released hemoglobin can be damaging tokidneys. Breakthrough due to lysis of erythrocytes is consideredimportant and drugs have been discovered and currently being used tocontrol intravascular hemolysis. This drug due to its downstream actiondoes not prevent extravascular hemolysis and therefore patients continueto remain anemic.

To prevent extravascular hemolysis from taking place; several majorcategories of complement inhibitors can be developed; a) Classicalpathway inhibitors that prevent C3b deposition produced via theclassical pathway onto the cell surface, b) Classical pathway inhibitorsthat prevent C3b formation produced via the amplification loop, c) APinhibitors that prevent the formation of CP derived C3b formation, andd) AP inhibitors that prevent C3b produced via the alternative pathwaywithout affecting the classical pathway. The inventor of the currentapplication claims those inhibitors that selectively target thealternative pathway derived C3b formation without affecting theclassical pathway derived C3b formation. The rationale for such anapproach is that such inhibitors would leave the C3b produced via theClassical pathway for host defense. The present inventors claim a genusof monoclonal antibodies that prevent the formation of C3b only via thealternative pathway without affecting the classical pathway derived C3b.

To prevent intravascular hemolysis; several major categories ofcomplement inhibitors can be developed; a) Classical pathway inhibitorsthat prevent C3b deposition produced via the classical pathway onto thecell surface, b) Classical pathway inhibitors that prevent C5b-9formation produced via the amplification loop, c) AP inhibitors thatprevent the formation of CP derived C5b-9 formation, and d) APinhibitors that prevent C5b-9 produced via the alternative pathwaywithout affecting the classical pathway. The inventor of the currentapplication claims those inhibitors that selectively target thealternative pathway derived C5b-9 formation without affecting theclassical pathway derived C5b-9 formation. The rationale for such anapproach is that such inhibitors would leave the C5b-9 produced via theClassical pathway for host defense. The present inventors claim a genusof monoclonal antibodies that prevent the formation of C5b-9 only viathe alternative pathway without affecting the classical pathway derivedC5b-9.

C3b and C5b-9 are produced via both the classical and the alternativepathways. The two C3 convertases (CP C3 convertase and AP C3 convertase)with different molecular structure have been identified; (C4b2a) and(PC3bBb). These C3 convertases cleave C3 and generate two differenttypes of C3b molecules. Since both complement pathways are independent,this invention only targets C3b production via the alternative pathwaywithout affecting the C3b produced via the classical pathway or from CPamplification loop. A genus of monoclonal antibodies that selectivelytargets the alternative pathway derived C3b are the focus of the currentinvention.

Antibodies of the present invention would control extravascularhemolysis in vivo and its associated clinical outcomes such as increasedreticulocite counts, hemoglobin (HgB) and LDH in clinical trials. Incertain embodiments, the present invention comprises a method oftreating a subject having hematological disorder wherein erythrocytes,neutrophils, monocytes, platelets and T lymphocytes are deficient in GPIlinked proteins, the method comprising administering an effective amountof an inhibitor that inhibits the alternative complement pathway toprevent the formation and deposition of C3b, PC3b, PC3bBb andP(C3b)n(Bb)n. Such an action is important for preventing extra- andintra-vascular hemolysis and episodes of hemolytic crisis. In otherembodiments, the invention comprises a method of treating a subjectpreviously treated with Eculizumab or a comparable drug wherein thesubject already is exhibiting extravascular hemolysis, the presentinvention is expected to dis-assemble to convertase and halt theprogression of extravascular hemolysis.

In certain embodiments, the methods of the present invention comprisetreating a subject having complement-mediated hemolytic disorderaffecting blood cells, wherein the subject exhibits at least one of thefollowing characteristics; a) the subject exhibits signs or symptomscontinued loss of red blood cells by ongoing or intermittentintravascular hemolysis and/or extravascular hemolysis; b) the subjecthas red blood cells opsonized by fragments of C3; c) the subjectrequires periodic blood transfusions; c) the subject has low normal orbelow normal levels of hemoglobin; e) the subject has low normal orbelow normal levels of platelets; f) the subject has high normal orabove normal reticulocytes; g) the subject has high normal or abovenormal bilirubin; h) the subject has iron overload or is at risk of ironoverload.

As preferred embodiments useful to accomplish the above methods, thepresent invention provides agents and compositions that inhibit theactivity of the complement alternative pathway. Such agents andcompositions comprise fusion proteins carrying the binding regions ofthe antibodies from the claimed genus and or antibodies themselves.These agents are expected to prevent the initiation of C3 convertaseformation and formation of C3b, prevent deposition of C3b onto cellsthat lack the GPI linked proteins. As a result, extravascular hemolysisis down-regulated, number of transfusions are reduced, cytopenia isreduced, and intravascular hemolysis is reduced. In another aspect ofthe present invention where reduction in cytopenia is claimed, cytopeniaincludes leukocytopenia, thrombocytopenia, erythrocytopenia,leukocytopenia, lymphocytopenia, and neutropenia. These processes canoccur as a result of cellular aggregate formation and removal of suchaggregates from subject's circulation. Reduction in cell number can alsooccur due to extravascular hemolysis.

In preferred embodiments, the inhibitor of the complement alternativepathway may comprise a fusion of the “Fab”, or a fragment comprising atleast the variable region of the antibody or a biologically activefragment thereof to the Fc region of the antibody. In another aspect ofthe current invention, the inhibitor of the complement alternativepathway may comprise only the blocking Anti-C3b antibody, Anti-Factor Bbantibody, Anti-Properdin antibody, and Anti-Factor D antibodiesspecially those that block both the formation of C3b and C5b-9. If suchantibodies block the formation of C3b and not the formation of C5b-9,then such antibodies are excluded from the current invention. Theselected antibodies of the genus should only inhibit the alternativepathway but not the classical pathway.

In a particular preferred embodiment, the inhibitor of the complementalternative pathway is a genus of neutralizing monoclonal antibodiesthat have the following characteristics:

Inhibit the alternative pathway derived C3b and do not inhibit theclassical pathway derived C3b. The classical pathway derived C3b isrequired for opsonization and for host defense. Thus the selected genusof the antibodies perform specific function.

These antibodies doe not inhibit the classical pathway and therefore donot inhibit the formation of C3b via the classical pathway.

The present invention provides in one aspect a method of treating asubject having a complement-mediated hemolytic disorder affecting bloodcells, the method comprising administering an effective amount of theantibody and its antigen binding fragments that inhibit activation ofthe complement alternative pathway, wherein the antibody inhibits theformation of both the C3b and C5b-9 responsible for extravascular andintravascular hemolysis respectively.

In certain embodiments of any of the methods described herein, thesubject has one or more of the following characteristics: a) the subjectexhibits signs or symptoms of cytopenia by ongoing or intermittentintravascular hemolysis and/or extravascular hemolysis; b) the subjecthas cell bound C3b wherein the cell is selected from the groupcomprising leukocytes, lymphocytes, erythrocytes, platelets, andmonocytes, basophils; c) the subject requires periodic bloodtransfusions; the subject has low normal or below normal levels ofhemoglobin; the subject has low normal or below normal levels ofplatelets; the subject has high normal or above normal reticulocytes;the subject has high normal or above normal bilirubin; or the subjecthas iron overload or is at risk of iron overload; or the subject hashigh number of dead cells.

In some embodiments, the method includes administering an effectiveamount of a monoclonal antibody selected from the collection ofantibodies of this invention which inhibit the activity of thecomplement alternative pathway and therefore;

Increase in the total number of cells to normal levels.

Increase the total number of surviving red blood cells increase tonormal levels.

Increase the total number of neutrophils to normal levels.

Increase the total number of monocytes to normal levels.

Increase the total number of T-lymphocytes to normal levels.

Increase the total number of platelets to normal levels.

Decrease the total number of dead cells, increase the total number ofhealthy cells.

Decrease the total number of cellular aggregates.

Decrease in the total LDH to normal levels.

Decrease bilirubin to normal levels.

Decrease hemoglobin in plasma.

Decrease in C3a, C3b, C5a, C5b, C5b-9, and sC5b-9 levels.

Decrease in activated cells.

Decrease in inflammatory cytokine levels.

Decrease in cellular activation.

In some embodiments, the antibodies of the current invention dampenand/or inhibit the activation of AP without inhibiting CP. They inhibitalternative pathway-dependent lysis and activation of cells involved ininflammation, inhibit production of inflammatory molecules andultimately inhibit a myriad of pathologies associated with varioushemolytic diseases. These antibodies also inhibit the formation of C3bresponsible for extravascular hemolysis and intravascular hemolysismediated via C5b-9.

C3b of alternative complement pathway (C3b)—This key molecule isimportant in the amplification of the alternative pathway. Whenassociated with a disease state, AP activation causes C3b to be producedand deposited on various cells, including blood cells. This, in turn,causes extravascular lysis of erythrocytes and platelets, the root causeof erythrocytopenia and thrombocytopenia respectively. For this reason,pathological overproduction of C3b is detrimental and must be inhibitedor controlled. C3b is also deposited on neutrophils, monocytes, andT-lymphocytes. Deposition of C3b on these nucleated cells does not causelysis of these cells but causes cell damage and equivalent to tissueinjury. C3b receptors are found on these nucleated cells and the densityof such receptors is increased during complement activation anddiseases. As a result, such cells become dysfunctional. It is theinvention of this application to demonstrate that not only erythrocyteswould be subject to lysis but also the platelets would be subject tolysis. Both erythrocytes and platelets are a nucleated cells. Thenucleated cells such as neutrophils, monocytes, and platelets—they allwould bind C3b and are removed via extravascular lysis.

We believe that if C3b formation is inhibited only via the alternativepathway, no C3b produced via the alternative pathway will be availablefor deposition on cells and therefore would inhibit cell loss viaextravascular lysis and/or removal by the host liver/or spleen.Inhibition of removal would mean cytopenia which includes all cells willbe inhibited. In hematological disorders such as PNH, not onlyerythrocytopenia is observed, but also thrombocytopenia, neutropenia,monocytopenia, lymphocytopenia is observed. It is important to addressremoval of leukocytes and platelets as these are new findings.

Neutrophils and other cells bear C3b receptors and therefore bound C3bcould be detected with the anti-C3b antibody. Neutrophils coated withC3b are incapable of fighting infections therefore the neutralizingmonoclonals of the claimed invention would prevent infection.

Similar to erythrocytes, platelets are also anucleated and thereforehave the ability to lyse.

C3b produced via the classical pathway is designated as (C3b) todifferentiate this C3b from those produced via the alternative pathway.C3b would remain intact for opsonization and removal of bacteria andtherefore must not be inhibited. The antibodies of the current genus donot inhibit the classical pathway and therefore do not inhibitassociated side products such as C3b, C3a, C5b, C5a, and C5b-9 producedvia the classical pathway. The process of opsonophagocytosis begins withdeposition of C3b on the surface of cells and the subsequent uptake byphagocytic cells. Inappropriate and/or uncontrolled production of C3bleads to inappropriate and/or uncontrolled opsonophagocytosis.

C3a—The C3a molecule is a peptide with a molecular weight of 9,000 Daand a high affinity for C3a receptors (C3aR). C3aRs are present onneutrophils, monocytes, platelets, mast cells, and T lymphocytes.Binding of C3a to C3aR activates the release of inflammatory moleculesfrom the triggered/activated cells. Upon activation, these cells: a)form intra- and inter-cellular aggregates, b) invade the normal tissueand host themselves causing pathology, and c) release inflammatorymediators such as TNF-α, IL_1, 11-18, IL-27, peroxides and proteasesthat can degrade the matrix and initiate inflammation and tissuedestruction. For example activated/triggered monocytes express CD11b andrelease Tumor Necrosis Factor alpha (TNF-α). Activated monocytes canform aggregates with platelets. Activated neutrophils also express CD11band release peroxides and neutrophil elastase. Activated plateletsexpress a higher concentration of CD62P and form aggregates withneutrophils and monocytes. Both mast cells and T lymphocytes are alsoactivated by C3a. C3a initiates the release of TNF-α from monocytes.TNF-α is known to play a key role in the pathological outcomes andconditions. Platelets also bear C3a receptors. Upon activation by C3a,platelets express CD62P, an activation marker. CD62P is responsible forinter cellular aggregate formation. These aggregates are removed fromcirculation, which ultimately leads to thrombocytopenia.

C5a also plays a role in activation of platelets. Regardless of themethod of platelet activation, activated platelets express CD62P, whichis also called P-selectin. P-selectin also mediates platelet-monocyteconjugation. This binding triggers the release of tissue factor frommonocytes.

C5a/C5b—AP C5 convertase (P(C3b)n(Bb)n) cleaves C5 and produces C5a andC5b. C5a is known to activate neutrophils and monocytes as C3aR and C5aRreceptors have been found on these cells. Upon activation, neutrophilsand monocytes produce inter and intracellular aggregates and releaseinflammatory markers such as neutrophils elastase, peroxides and avariety of matrix proteases that degrade the tissue matrices. Similar toC3a, C5a also causes the release of inflammatory mediators relevant toseveral pathologies and associated hemolytic diseases.

C5b-9 and sC5b-9—These complexes are also called “MAC”, the C5b-9 is acomplex that forms on the cell surface and causes tissue injury. Asdemonstrated in FIG. 6, rabbit erythrocytes (rRBC) activate the AP inwhole blood. In response, C5b-9 is integrated in the cell membrane,causing lysis of these cells by human complement. This assay representsa way of demonstrating tissue injury using an erythrocyte hemolysisassay. The sC5b-9 is a MAC complex that is formed by the association ofprotein S to C5b. C5b binds S instead of depositing on a cell surface.Protein S enables the formation of “soluble MAC,” abbreviated as sC5b-9.Soluble MAC also activates platelets and other cell types.

C3a and C5a activates cells, activated cells express markers such asCD62P and CD11b. These activated cells form aggregates. Aggregates areremoved from circulation leaving patient cytopenic.

The antibodies of the present invention can prevent AP derived formationof C3a, C3b, C5a, C5b, and C5b-9. As a result, cellular activation isprevented. If there is no activation, there is no release ofinflammatory markers. Thus, the antibodies of this invention are capableof blocking, preventing the progression of the disease.

Role of Alternative Pathway in Hemolytic Diseases

Based on the available literature and associated data, it appears thatin chronic hemolysis, complement activation is mediated predominantlyvia the formation of C5b-9 on cell surfaces. It does not differentiatebetween the classical pathway derived or the alternative pathwayderived. This invention targets the C5b-9 formed via the alternativecomplement pathway, but not the classical complement pathway. Thisinvention would leave the classical pathway intact for host defenseagainst infection.

Hemolytic diseases include those in which lysis of erythrocytes resultsin a release of hemolglobin. Such actions reduce the total concentrationof erythrocytes in the blood. Paroxysmal nocturnal hemoglobinuria(“PNH”) is a rare hemolytic disease. It is an autoimmune disorder of theblood wherein erythrocytes are destroyed by activities of the body's owncomplement pathways. PNH results from somatic mutations which rendercells unable to synthesize the glycosyl-phosphatidylinositol (“GPI”)anchor. The GPI anchor protects cells against complement attack. PNHcells are deficient in complement-regulating surface proteins thatinclude the decay-accelerating factor (“DAF”), or CD55, and membraneinhibitor of reactive lysis (“MIRL”), or CD59.

In PNH, lysis of erythrocytes causes a pathologic reduction in the totalerythrocyte count (i.e., hemolytic anemia). The presence of hemoglobinin the urine (hemoglobinuria) is particularly evident after sleeping andusually causes the urine to appear dark in color. Subjects with PNH willalso have free hemoglobin in their bloodstream (hemoglobinemia).Hemolytic anemia is due to intravascular lysis of red blood cells bycomplement component C5b-9 (MAC). Reduced numbers of erythrocytes andplatelets cause dysphagia, fatigue, erectile dysfunction, thrombosis andrecurrent abdominal pain.

Erythrocyte Lysis—Erythrocytes are anucleated cells and are responsiblefor maintaining the hemoglobins. These cells are known to be subject tocomplement attack in PNH due to the absence of CD55 and CD59 from thecell surface. These cells are therefore subject to C3b deposition andremoval via extravascular lysis. These CD59 deficient cells also allowdeposition of C5b-9 and erythrocyte removal via intravascular lysis.Lysis results in hemoglobin release from these erythrocytes causinghemolytic anemia and therefore decrease in the number of erythrocytes ingeneral causing erythrocytopenia. Thus erythrocytes are subject toremoval via both extra- and intra-vascular lysis. Additionally,excessive free hemoglobin can cause kidney damage and system loss ofiron. Haptoglobin helps ameliorate the situation by binding freehemoglobin and facilitating enzymatic degradation of the boundhemoglobin.

Pathologic intravascular hemolysis, such as that associated with PNH andother hemolytic diseases, often results in concentrations of freehemoglobin high enough to completely deplete haptoglobin. Oncehaptoglobin has been depleted, the burden is then on the kidneys tore-absorb the free hemoglobin. Once the kidneys reach their capacity forhemoglobin re-absorption, hemoglobinuria begins. The release of freehemoglobin during intravascular hemolysis results in excessive oxidationof nitric oxide (NO) to nitrate (NO₃₋) The depletion of NO causesenhanced smooth muscle contraction, vasoconstriction and plateletactivation and aggregation. The systemic consequences of excess freehemolglobin in blood also effect abdominal pain, erectile dysfunction,esophageal spasm, and thrombosis.

As a routine laboratory test, blood smears are, generally, evaluated toidentify morphologic abnormalities of RBCs (Red Blood Cells),reticulocyte count (to determine bone marrow compensation for RBC loss),lactate dehydrogenase (LDH), and levels of free hemoglobin (fromhemolysis). Concentrations of bilirubin, haptoglobin, hemosiderin, andfree hemoglobin can measure the extent of hemolysis and helpdifferentiate between intravascular vs. extravascular hemolysis. RBCnumbers, levels of RBC (i.e., cell-bound) hemoglobin, and hematocrit areoften evaluated to determine the extent of any anemia and/or any otherassociated symptom of hemolytic disease. Levels of Lactate Dehydrogenase(LDH) can also provide some information with regards to the extent ofongoing cell death.

Lysis of erythrocytes sometimes could give erroneous and inconsistentresults due to persistent extravascular and intravascular hemolysis.Therefore cells that do not undergo lysis would be better fordetermining the clone size in PNH patients. Such examples areneutrophils and other mononuclear cells.

Convertase Laden Erythrocytes—In PNH, erythrocytes that lack the CD55,would be prone to C3b deposition. Such cells not only have the C3b butalso have the entire C3 convertase. Antibodies of the current genus,prevent the formation of C3b, C3bBb, PC3bBb formation and thereforewould prevent the lysis of erythrocytes via extra- and intra-vascularhemolysis. Thus these antibodies would prevent the formation of both theC3 and C5 convertases.

Antibodies of the current invention, those that selectively prevent theformation of C3b and C5b-9 produced via the alternative pathway wouldinhibit extra- and intra-vascular hemolysis with resultant benefit intotal anemia. Platelet Lysis—Platelets are anucleated and thereforesubject to complement attack via the alternative pathway. Similar toerythrocytes, platelets are also destroyed via the similar mechanism.Lysis of platelets would occur in PNH patients where platelets lack theCD55 and CD59 on its cell surface. Platelet lysis means reduction inplatelet number and therefore blood clotting ability of blood in PNHpatients. The reduction in platelet number results in increased levelsof platelet contents including but not limited to platelet factor 4(PF4), platelet derived growth factor (PDGF), beta thromboglobulin,P-selectin. This includes all contents that are reported now or infuture are covered under this invention. Thus antibodies of the currentinvention would decrease thrombocytopenia associated with patients withhematological disorders.

Lysis of Nucleated Cells—Under this category fall cells such asNeutrophils, monocytes, and lymphocytes. These cells are known to beCD55/CD59 positive and have recently been considered reliable cells forestablishing PNH clone. Often higher percentage of leukocytes aredetected with CD59 than shown with erythrocytes. Erythrocytes generallyhave a lower life span compared to leukocytes. Nucleated cells do notlyse and therefore are present in blood for longer duration compared toerythrocytes and therefore are more confirmed markers of PNH.

Neutrophils bear C3b receptors and therefore would bind such molecules.We show increased staining of C3b, properdin, and Bb on neutrophilsindicating that the convertase forms on such cells. Antibodies of thecurrent invented genus of antibodies is capable to preventing theformation of alternative pathway derived C3 convertase but not classicalpathway derived C3b. Similar finds have been noted on all nucleatedcells. It was surprising to note that nearly all nucleated cells showedheavy staining with both C3b and C5b-9. Both of these molecules depositon cell surface as a result of AP activation.

It is the intent of the proposed invention to prevent the formation anddeposition of C3b and C5b-9 on the nucleated and non-nucleated cells. APspecific selected antibodies inhibit the pathway upstream and preventthe deposition of both molecules that cause extra and intravascularlysis and damage. Nucleated cells when laden with C3b and C5b-9 arelikely to become dysfunctional and recognized lend themselves to death.Dead cells are recognized by the stain specific for cell death.

Role of C3a and C5a in Inflammation: Elevated levels of C3a and C5a arepredicted in PNH due to the continuous activation of the alternativepathway leading to lysis of erythrocytes. C3a and C5a have potentpro-inflammatory and immuno-regulatory functions. They increase vascularpermeability and serve as chemo attractants, which promote soft tissueswelling. The anaphylatoxins activate neutrophils and monocytes, whichresults in the production of pro-inflammatory mediators such as TNF-αIL-1, IL-6, IL-8, and IL-17 [47-50]. C5a is a potent chemotactic proteinthat induces neutrophil chemotaxis, de-granulation, neutrophil elastaserelease, and superoxide generation. Neutrophils contain a potent arsenalof vasoactive, proteolytic and cytotoxic substances, which are producedto mediate many of the manifestations of inflammation and cellular lysisin hemolytic diseases such as PNH. Compounds of the current inventioninhibit the detrimental inflammation, tissue injury, and cellular lysis.

Blood Transfusion and Anti Complement Antibodies—

Blood cell transfusion is given when the patient has too few red bloodcells (anemia). Blood tests in PNH show changes consistent withintravascular hemolytic anemia: low hemoglobin, raised lactatedehydrogenase, raised reticulocytes (immature red cells released by thebone marrow to replace the destroyed cells), raised bilirubin (abreakdown product of hemoglobin), and decreased levels of haptoglobin.Anemia causes weakness and tiredness. In severe cases, it can causeshortness of breath or a rapid heartbeat. Transfusions are usually usedwhen the hemoglobin level is less than 8 grams per deciliter. Sometimesinstead of a transfusion, you may get a red blood cell growth factor—adrug that helps your body make more red blood cells. This growth factoris called erythropoietin (Procrit®, Epogen®, Aransep®). Platelet,another anucleated cell would also decrease in number based oncomplement-mediated lysis. Doctors prescribe platelet transfusions tokeep the platelet count above 10,000 to 20,000 (per cubic millimeter).Transfused platelets last only two to three days. The antibodies of thecurrent invention are expected to preserve the added platelets andprevent the destruction of platelets made by the patient's body.

White blood cell (granulocyte) transfusions are rare. This is becausethe granulocytes last only a few hours in the bloodstream. Donated whiteblood cells must be used right away and do not last long. A commonexample is filgrastim (Neupogen®) for increasing the number ofneutrophils/leukocytes. The antibodies of the claimed genus can helpprevent the damage and lysis of cells that are increased by theadditives.

To prevent cytopenia in general, the antibody of the claimed genus couldprevent C3b formation and deposition and C5b-9 formation and deposition,the two main functions of the AP derived moieties are important forhemolytic disorder whether it is with or without the additives.

Role of Complement System Activation in PNH and Other Hemolytic Diseasesand Conditions

Elevated levels of C3a, C5a, C3b, C5b, and C5b-9 can gauge the level ofactivation, inflammation and hemolysis in disease conditions. Examplesof complement-associated disorders involving hematologic disordersinclude, but are not limited to: Catastrophic anti-phospholipid syndrome(CAPS), Cold Agglutinin Disease (CAD), which increases c3b, Thromboticthrombocytopenic purpura (TTP), which increases CD46, factor H, andfactor I, Idiopathic thrombocytopenic purpura, where C3 and C4 detectedare on platelets, Serum sickness, where abnormal factor H leads toincreased glomerular C3 deposition, Endotoxemia, Sepsis, Atypicalhemolytic uremic syndrome (ahus), where there is enhanced formation ofc3bbb convertase and resistance to complement regulators, ParoxysmalNocturnal Hemoglobinuria (PNH), where it has been shown a C5 antibodytreatment reduced thromboembolism risk, Septic shock, sickle cellanemia, which elevates c3b, Hypereosinophilic syndrome, which increasesc5a, anti-phospholipid, Autoimmune lymphoproliferative syndrome, Dego'sdisease, where c5b-9 is activated, Evan's syndrome, essential mixedcryoglobulinemia, and pure red cell aplasia. Antibodies of the inventiongenus of antibodies, selected with The Screening Method (see Page 23),can prevent local damage and have shown benefit in whole blood models ofthe disease. Antibodies of the current invention prevent bloodinflammation and cellular lysis and the associated maladies.

Classical Pathway Versus Alternative Pathway C3 Convertases and PNH

Both the classical and the alternative pathway C3 convertases areresponsible for the cleavage of central C3. Eculizumab inhibits both theCP and the AP at the C5 level and does not inhibit the formation of C3aand C3b. This is a severe disadvantage of Eculizumab since both excessC3a and excess C3b have been implemented in several disease conditions.C3b produced by both, or either, pathways can coat cells. These cellscan then be removed via the opsonization process.

Conceptually, inhibition of the AP, but not the CP, will allow for CPdependent production of C3b which may be required, in case of infection,for opsonization via the CP. The antibodies of the invention genus ofantibodies only inhibit the AP. They do not inhibit the CP or anyamplification loop of the CP. Inhibition of C3b is essential to preventextravascular lysis (and effective removal) of erythrocytes, while CPdependent C3b is essential to host defense. In diseases such as PNH, itis the alternative pathway that is problematic, not the classicalpathway. Thus, to combat overproduction of these proteins, the besttreatment is to shut down the alternative pathway alone. Presentthinking on the subject is that the two pathways and inextricablyconnected, and that it may not be possible to shut down the AP withoutinhibiting the CP. The present invention presents a challenge to thepresent thinking and offers a method for shutting down the AP without,to any degree, inhibiting the CP. Any treatment that shuts down orsignificantly inhibits the classical pathway will jeopardize the body'sability to fight infection. C3b production is needed for the removal ofunwanted cells, such as infectious bacteria. Therefore, it is desirableand advantageous to preserve production of CP dependent C3b whileinhibiting AP dependent C3b.

Selection and Identification of the Claimed Genus of Antibodies

While both the classical and alternative pathways produce C3a and C5a,the present invention selectively inhibits AP produced C3a and C5a.Equimolar concentrations of C3a and C3b are produced as a result of theC3 cleavage. Thus inhibition of C3b formation in vitro can bedemonstrated by the assays described herein. The formation of C3bevidences the concurrent formation of C3a (and vice versa). Anyantibody, targeting a component of the C3 convertase complex, which actsto inhibit the cleavage of C3 into C3b and C3a, will inhibit C3b and C3ain equal measure.

Inhibition of C3a production will also inhibit all of the activities ofC3a. Such activities include: subsequent activation of neutrophils,monocytes, platelets, basophils, and T lymphocytes, as well asproduction of inflammatory markers. (See FIGS. 13 through 18.) C3bdeposits on the cell surfaces via C3b receptors. C3b deposition isrequired for opsonization/removal of erythrocytes and other cells whichcause pathological outcomes in other hemolytic diseases

U.S. Pat. Nos. 6,333,034 & 7,423,128 claims antibodies that inhibit bothCP and AP mediated complement activation and therefore host defense iscompromised. These antibodies play in role how antibodies prevent theformation of properdin oligomer. Properdin is a thrombospondin type 1repeat and consists of six repeats of thrombospondin type1. Theseantibodies inhibit the binding of properdin to C3b and prevent theformation of C3c. C3b cleavage results in the formation of C3c. Thusthese antibodies prevent the cleavage of C3b.

In another aspect, the alternative pathway specific antibody of thepresent invention can bind to the alternative pathway protein withoutreducing the levels of that protein in the human.

Therapeutic Antibodies

Referring to FIG. 1, we show that both CP and AP are distinct and notconnected. It is known that CP has an amplification loop and thatconnects the CP and the AP. The schematic only shows how the antibodiesof the current genus work and not the way antibodies of other inventionswork. AP amplification is shown in the upper right hand side andconsists of PC3b, PC3bB, PC3bBb. As can be seen in the schematic, PC3bBbthen acts to perpetuate the cycle by cleaving C3 into more C3b whichbinds to P to again form PC3b. Application of the antibodies selectedusing the screening method described herein completely inhibits thealternative pathway without affecting the classical pathway byspecifically targeting the components of this amplification loop. Theseantibodies prevent the amplification loop of the alternative complementpathway without affecting the classical pathway (as shown on the leftside of the schematic in FIG. 1).

Based on the old conversion of pathway at C3 theory, those with ordinaryskill in the art would expect any activation of the classical pathway toinvariably have the effect of triggering and propagating the alternativepathway. This is because the two pathways are believed to “overlap” atthe starting point of the C3. According to this theory, C3b produced viathe classical pathway participates in the AP amplification loop. Thegenus of antibodies selected using the method described hereinspecifically targets components of the alternative pathway amplificationloop in such a way as to inhibit the alternative pathway regardless ofwhether or not the AP amplification loop has been otherwise triggered bythe classical pathway. Thus, for example, anti-C3b antibodies describedherein only inhibit the AP and not the CP amplification loop or the CPpropagation.

The “Screening Method”: Selection of Antibodies that Inhibit theAlternative Complement Pathway, do not Inhibit the Classical ComplementPathway, and are Specific for Components of the AP C3 Convertase

Antibodies specific for complement proteins belonging to the alternativepathway (whether part of the CP amplification loop or an alternativepathway by itself), such as C3b, P, Ba, and Bb can be screened using a“Screening Method” described herein to select antibodies to inhibitalternative complement pathway without affecting the CP or theamplification loop of the CP. C3b, P, Ba, and Bb are large proteins of,respectively, 210K, 50K, 33K, and 66K molecular weight. One skilled inthe art can generate millions of antibodies to each of these proteins.Production of antibodies to a target is, by itself, meaningless withoutfurther selection of those antibodies as having a specific therapeuticfunction.

In some embodiments, the Screening Method can include a two-stagescreening process. The first stage utilizes three successive screeningassays to identify Type AP antibodies (antibodies which specificallyinhibit the AP). The selection process leads to identification ofalternative pathway specific antibodies which are similar infunctionality but targeted to a wide variety of antigens. These selectedantibodies cannot be differentiated based on the targets they bind to orthe species of animals in which they were raised. Upon sequencing, it isclear that such antibodies have widely different CDRs (the regionsinvolved in binding to antigens). The functionality, and ultimatetherapeutic value, of these antibodies can't be defined by theirsequences alone. The Screening Method described herein can identify anddefine a genus of antibodies against Properdin (P), Factor C3b (C3b),and/or Factor B (Ba, or Bb) which have the desired functionality andeffect.

A1) Step 1: Selection Based on Function

Three distinct types of antibodies can be identified using specificassays. The antibodies can be referred to as Type CP, Type CP/AP, andType AP, and are defined as follows.

Type CP: These inhibit the classical pathway but not the alternativepathway.

Type CP/AP: These inhibit both the classical pathway and alternativepathway.

Type AP: These inhibit the alternative pathway but not the classicalpathway.

Identification of these three different types of complement inhibitingantibodies is accomplished using three different assays; a CP onlyassay, a combined CP and AP assay, and an AP only assay.

Type CP

For classical pathway activation, antibody sensitized sheep cells areused as an activator in 1% normal human serum in the presence ofCa2+/Mg2+. The calcium ions are required for the activation of theclassical pathway for the initial trigger of the C1q/C1r/s complexes. CPwill not occur in the absence of the Calcium ions. Mg2+ is required foralternative pathway activation. In 1% normal human serum containingCa2+/Mg2+, only the CP proceeds to completion. Without the requisitelevels of NHS which is 10%, the alternative pathway will not have asignificant presence. FIG. 2, Assay-1 shows that shows that CPactivation leads to the lysis of antibody sensitized sheep red bloodcells. Antibody bound sRBCs act as a trigger for the classical pathway.The observed CP activity is isolated from AP activity by using a 1%buffer solution containing Ca2+ and Mg2+. FIG. 6 also shows that none ofthe selected antibodies materially inhibit CP mediated hemolysis of thesRBCs in 1% human serum in classical pathway condition.

Type CP/AP

For classical pathway activation, along with the amplification loop ofthe alternative pathway, antibody sensitized sheep red blood cells areused as an activator in 10% Ca2+/Mg2+ in normal human serum. Thedifference between the assays used to identify Type CP antibodies andthose used to identify Type AP/CP antibodies is the concentration ofnormal human serum which is 10% in Type CP/AP. The concentration ofCa2+/Mg2+ used in the identification of Type AP/CP antibodies, byproviding the level of Mg2+ required for AP activation, allows for boththe CP and the AP to be active. Antibody sensitized sRBCs only activatethe CP. They do not, by themselves, activate the Alternative Pathway.However, in the presence of sufficient NHS, activated CP will utilizethe amplification loop of the AP. Thus, the assay system is designed toevaluate the performance of complement inhibitors under conditions inwhich both pathways are active (FIG. 3, Assay 2 and FIG. 4 Panel B).Under these conditions, the C3b produced via the classical pathway canfeed into the alternative pathway causing “amplification of thealternative pathway loop” of the alternative pathway and can serve as atrigger indirectly. In other words, the AP has been activated by the CP.Antibodies that prevent CP initiated activation of the AP have beendescribed in (R Gupta-Bansal, J B Parent, K R Brunden, Molecularimmunology. 37(5):191-201). These antibodies reduced hemolysis of thesheep red blood cells in these assays. However, at this stage in The“Screening Method”, the antibodies have yet to be differentiatedaccording to how (and where) they inhibit the process. Antibodies whichinhibit the classical pathway's activation of the AP by inhibiting anystage of the CP are not included in the selected genus of antibodies.Accordingly, the antibodies that inhibit the classical pathway initiatedamplification of the alternative pathway have been excluded from theselected genus of antibodies.

Type AP

Rabbit RBCs (rRBC) are used to activate the AP in 10% normal human serumin the presence of Mg2+ and in the absence of Ca2+. Because the CPrequires the presence of Ca2+, the classical pathway will not be activeunder these conditions. Thus, in 10% NHS in Mg2+, only the AP proceedsto completion. As shown in FIG. 2, Assay-3, AP activation leads tocellular lysis of the rRBCs. It should be noted that this assaydemonstrates that the alternative pathway can be activated, and progressto completion, in the absence of active classical pathway function. TheAP does not require initiation by the classical pathway in order toproceed. FIG. 4 clearly shows that the invention genus of antibodiesinhibits AP dependent hemolysis of rRBCs in 10% normal human serum.

When an antibody's effect on AP activation and progression is observedin isolation, with the AP as a stand-alone process, the informationobtained is different than the information obtained from observation ofthe antibody's effects in conditions where both the CP and the AP areactive. The information obtained here is also different than thatobtained from observation of the antibody's effect in conditions whereonly the CP is active.

Analysis of the Three Assays

If the presence of a particular antibody(s) in one of these three assayswas found to reduce the rate of hemolysis, it was concluded that thatantibody inhibits the pathway, or pathways, which were active in thatassay. Thus, for example, if an antibody was found to reduce hemolysisin all three assays, it was concluded that the antibody inhibited boththe AP and the CP (Type CP/AP). If an antibody was found to inhibithemolysis only in assays containing 1% human serum (with Ca2+/Mg2+buffer) it was concluded that that antibody inhibited the CP but not theAP (Type CP). If an antibody was found to inhibit hemolysis only in theassays containing 10% human serum and Mg2+(but not Ca2+), it wasconcluded that the antibody inhibited the AP but not the CP.

This is the first stage of The Screening Method. Antibodies passingthese selection criteria have been shown to: 1) inhibit the alternativepathway under conditions in which the alternative pathway is active inisolation (i.e., without concurrent activation of the classicalpathway), and 2) have no effect on CP activity (either in isolation orwhen concurrent with AP activity).

The invention uses this combination of assays to first identify Type APantibodies. However, additional screening steps are needed in order toidentify the selected genus of antibodies. Additional screening isnecessary because these assays will identify antibodies in both theupstream and the downstream portion of the AP and the CP.

A2) Step 2: Selection of Those Type AP Antibodies which Act on C3Convertase Formation

The second step of the Screening Method is to identify which Type APantibodies inhibit only the functional activity of AP C3 convertase. Inother words, this step identifies those Type AP antibodies which act“up-stream” of the alternative complement pathway system, at theamplification loop of the AP, rather than “down-stream.”

This step is accomplished by first establishing a solid phase ELISAbased binding assay. This assay allows for the direct detection of C3band C5b-9 produced via the alternative pathway. Detections of theseproteins represent an early component (C3b) and a late component (C5b-9)of the alternative complement pathway. If an antibody inhibitsproduction of C5b-9 but not C3b, it is likely to be acting on the C5convertase of the AP. By contrast, C3b production will be inhibited byantibodies that inhibit the activity of the C3 convertase. Inhibition ofC3b production will also inhibit production of C5b-9 (because C5b-9 isproduced downstream of C3b). Thus, Stage 2 of The Screening Methodseparates antibodies inhibiting C3 convertase (up-stream) from thoseinhibiting C5 convertase (down-stream).

The Screening Method identified antibodies that selectively inhibit theAP C3 convertase. This stage of selection utilizes an assay in whichhuman serum at 10% in the presence of Mg++ is allowed to incubate overan LPS coating. LPS is a specific activator of the alternative pathwayand can allow formation of AP derived C3 convertase and C5 convertase.As shown in FIG. 6, the selected genus of antibodies prevents theformation of C3b, a central component of the alternative pathwayamplification loop. FIG. 7 shows that they also inhibit formation ofC5b-9.

At the conclusion of the this stage selection process, antibodies thatprevent the AP dependent cellular lysis and C3b formation (FIG. 6) areselected as being members of the selected genus of antibodies. Theseantibodies are defined by the fact that they all: 1) selectively inhibitthe alternative pathway without inhibiting the classical pathway (FIGS.2, 4, and 5), and 2) inhibit the alternative pathway dependent C3bformation, by acting on C3 convertase formation, an upstream componentof the AP.

FIGS. 10 and 11 show that the selected genus of antibodies inhibitsformation of complement proteins C3a and C5a, respectively.

Selected Genus of Antibodies

Application of the Screening Method has thus far produced severalantibodies from the selected genus of antibodies.

C3b as Target Protein

Mouse Anti Human C3b (Anti-C3b)

C3b is a large protein and therefore multiple antibodies can be producedagainst various segments of this protein. There exist multiple siteswhere-on an antibody might bind and inhibit the protein's activity inany variety of ways. Depending on how and where an antibody binds toC3b, the effect of that antibody could range from inconsequential tocomplete inhibition. Injecting a mouse with Human C3b will result in theproduction of a myriad of mouse antibodies against the Human C3bprotein.

The selected genus of antibodies include those that bind to C3b in sucha way as to prevent the interaction of C3b with Factor B. The effect ofthese antibodies is necessarily isolated to the alternative pathwaysince no such interaction exists within the classical pathway. Theseantibodies prevent the formation of C3a/C3b, C5a/C5b, and C5b-9/sC5b-9critical for pathological outcome causing disease initiation andprogression. Inhibition of the formation of each of these molecules, bythe alternative pathway, has significant physiological consequences.Inhibition of alternative pathway produced C3b (herein referred to as“aC3b”) impacts extravascular hemolysis of erythrocytes. The C3bproduced by the classical pathway is not inhibited by these antibodiesand therefore is required for opsonization of foreign particles/bacteriathat are coated with CP produced C3b (herein referred to as “cC3b”).Thus, the selected genus of antibodies prevents the formation of aC3band not cC3b by such antibodies that have this as a common function. Theinhibition of C3a formation has direct effect on monocytes activationand production of TNF-α which is a validated marker of inflammation.

Properdin as Target Protein

As is the case with C3b, Properdin is a large protein with manypotential sites where antibodies can bind. Different antibodies bindingin different ways and/or on different sites of the Properdin protein,will inhibit either amplification loop of the classical pathway oralternative pathway. Properdin is known to be part of the amplificationloop of the classical pathway. Thus, classical pathway activation can bedampened by the use of specific anti-properdin antibodies that inhibitthe amplification loop (U.S. Pat. No. 6,333,034). Some antibodies caninhibit the classical pathway activation where interactions of Properdinto C3b, within the classical pathway, become important for classicalpathway amplification. (U.S. Pat. No. 6,333,034)

Properdin binds to itself and generates aggregates. Depending upon theconfiguration of the aggregate, antibodies binding Properdin can bindmono, di-, tri- and tetramer, with each generating different responses.Thus antibody-to-properdin ratio can be 1:1, 1:2, 1:3, and 1:4. Thismeans that an antibody can bind in any configuration. An assay can beused to separate antibodies in a rank order according to potency, by theratio at which they bind Properdin. In other words, antibodies that bindat a 1:1 ratio can be separated from those that bind at a 1:2 ratio, a1:3 ratio and a 1:4 ratio. A binding ratio of 1:1 suggests that theantibody binding is via one arm and not by two arms. Such antibodiesdemonstrate a 1:1 binding ratio regardless of whether or not theantibody is a Fab (monovalent) or the IgG (divalent).

Properdin is involved directly in the AP activation but indirectly inclassical pathway activation via the amplification loop in vivo. Also,Properdin binds both C3b and C5b. An antibody which disrupts Properdin'sinteraction with C3b may or may not interrupt Properdin's interactionwith C5b (and vice versa). Antibodies that prevent one or both may be ofdistinguishable clinical significance.

Thus, some antibodies targeting Properdin a) inhibit both the classicalpathway and alternative pathway, or b) inhibit the alternative pathwayalone. The selected genus of antibodies would only include thoseantibodies targeting Properdin which acted on Properdin in specificallysuch a way as to only inhibit the AP, and not the CP.

Anti Human P (Anti-P) Derived from Mouse

The protein Properdin (P) is a large protein with a molecular weight ofapproximately 50,000. A multitude of antibodies can be produced againstvarious protein motifs of this large protein. Not all, or even most, ofthese antibodies will necessarily have any therapeutic value.Identification and selection of the appropriate antibody, or antibodies,those with optimal therapeutic value, is crucial.

Two mouse anti-human-P antibodies were selected using a proprietarycombination of successive screening methods. The Screening Methodenabled this inventor to identify those antibodies which 1) bind tohuman Properdin, 2) selectively inhibit only the activity of thealternative complement pathway, and 3) interrupt the alternative pathwayin such a way as to not disrupt the amplification loop of the classicalpathway. These antibodies bind properdin as the target antigen. And theydo so in such a way as to inhibit the formation of the P(C3b)n, PC3bBand PC3bBb, and by extension, (Bb)n and C3bBb. The inhibition of thesespecific complexes is one of the essential and defining commoncharacteristics of all the antibodies of the selected genus. Inpreventing the formation of these complexes, these antibodies allprevent the alternative pathway's production of C3b, C5a, C5b, andC5b-9, as well as TNF-α, IL-1.

Anti Human P (Anti-P) Derived from Rabbit

Three rabbit anti-human-P antibodies were selected using the ScreeningMethod (the same that was used for selecting the anti-human-P mouseantibodies). As can be seen in FIG. 2, these antibodies inhibit thealternative pathway dependent lysis of rabbit red blood cells (rRBC) innormal human serum (NHS) in buffer that lacks calcium and thereforethere is no contribution from the classical complement pathway. In doingso, the effect of these antibodies is targeted, and isolated, to asection of the alternative pathway which does not overlap with theclassical pathway. These antibodies prevent the formation of C3a, C3b,C5a, C5b, and C5b-9. The formation of these specific proteins is thecritical step in the alternative pathway wherein a normal immune systemprocess can become the source of a pathological condition. It's theoverproduction of these proteins from the alternative pathway that oftencause arthritic conditions.

The selected alternative pathway specific anti-human-P antibodiesgenerated in rabbits are analogous in effect to those from the mousemodels. They are analogous in effect because both the mouse and therabbit derived antibodies were selected using The Screening Method. Theyinhibit the formation of C3a, C3b, C5a, C5b, and C5b-9; therebyinhibiting the activation of monocytes, neutrophils, platelets, and theformation of TNF-α (which also plays a key role in inflammation).

Sequences of these anti-properdin rabbit antibodies are very differentas shown in the tables noted below. Therefore, looking at the proteinsequences alone would not necessarily yield any understanding of theireffect on Properdin. Unless tested using the Screening Method, it wouldbe difficult to determine if a given antibody belongs to the selectedgenus of antibodies. Accordingly, the selected genus of antibodies can'tbe defined by a specific amino acid sequence. Rather, the genus isdefined by the ability of its member antibodies to 1) selectivelyinhibit AP activation without disrupting any function of the CP, and b)act on a specific part of the AP that is isolated from the CP and whichis responsible for AP production of C3a, C3b, C5a, C5b, and C5b-9.

Ba as Target Protein

Anti Human Ba (Anti-Ba) Derived from Mouse

The protein Ba (cleaved from Factor B) is a large protein with amolecular weight of approximately 33,000. Thus, like Properdin and C3b,any of a multitude of antibodies can be produced against various proteinmotifs of, and locations on, the protein. With this protein, as with theother proteins of the AP, the invention is a selected genus ofantibodies which bind to the protein in such a way as to inhibit theformation of C3a, C3b, C5a, C5b, and C5b-9, which are required for thepathological progression of the disease.

As can be seen in FIG. 2, these antibodies inhibit the alternativepathway dependent lysis of rabbit red blood cells (rRBC) in normal humanserum (NHS) in a buffer that lacks calcium. The classical pathway can'tfunction in a buffer which lacks calcium. Thus, in these conditions,there is no contribution from the classical complement pathway. Suchconditions enable one to observe the effect these antibodies have on thealternative complement pathway in the complete absence of the classicalpathway. Observing the antibodies under these conditions is one step ofthe Screening Method by which the antibodies of the invention areidentified.

The sequencing of these anti-alternative pathway antibodies are verydifferent. Thus, here again we observe that the selected genus ofantibodies can't be defined by a specific amino acid sequence. Rather,the genus is defined by those which are selected by The ScreeningMethod.

Bb as Target Protein

Anti Human Bb (Anti-Bb) Derived from Mouse

The protein Bb (cleaved product of Factor B) is a large protein with amolecular weight of approximately 64,000. Thus, where again we find thatseveral antibodies can be produced against various protein motifs ofthis protein. Again we apply the Screening Method in order to produceonly those antibodies which have the desired effects. Mouseanti-human-Bb antibodies were raised against factor Bb and thereforewould not bind the Ba fragment of the antibody. These monoclonalantibodies were also selected using the Screening Method. They bind Bband factor B, but not Ba as the target antigen. The selectedanti-human-Bb antibodies share the features characteristic of theselected anti-C3b, anti-P, anti-Ba antibodies. Like all of theantibodies from the selected genus of antibodies, these anti-human-Bbantibodies prevent the formation of C3a, C3b, C5a, C5b, and C5b-9 by thealternative pathway. In so doing, these antibodies also prevent theformation of well known markers of inflammation such as TNF-α, IL-1.

Anti Human Bb (Anti-Bb) Derived from Rabbit

Three rabbit anti-Human Bb antibodies were selected using the ScreeningMethod. Members of the selected genus of antibodies which bind Bb do notalso bind Ba. Factor B is an integral component of the alternativecomplement pathway but not the classical complement pathway. Antibodiesbinding human Bb, which survive The Screening Method, prevent theformation of complexes critical for the propagation of the alternativepathway; C3bB, PC3bB, C3bBb, PC3bBb, P(C3b)n(Bb)n. Like all antibodiesof the invention, they prevent the AP induced formation of C3b, C3a,C5b, C5a, and C5b-9, and inhibit the AP at a juncture not shared withthe classical pathway. Inhibition of formation of each of thesemolecules has physiologic consequences. Inhibition of C3b (aC3b) willimpact extravascular hemolysis. Inhibition of C3a and C5a will impactcellular activation and subsequent release of inflammatory mediators.Inflammatory mediators, when over-produced, can cause any number ofdisease pathologies in humans.

As with other antibodies of the selected genus, sequences of theserabbit anti-Bb antibodies are very different. Therefore, looking at theprotein sequences alone would not enable one to predict whether suchantibodies could have the desired effect.

Table 1 and Table 2 list the amino acid sequences of the heavy and lightchains of anti-C3b, anti-P, ant-Ba, and anti-Bb antibodies that wereselected using the Screening Method described herein. The Tablesidentify the heavy chain and light chain CDR1s, CDR2s and CDR3s of theantibodies as well as in the respective frameworks. Accordingly, aspectsof the application described herein, relate to an isolated monoclonalantibody, or antigen binding portion thereof comprising: (a) a heavychain variable region comprising CDR1, CDR2, and CDR3, of the respectiveantibodies; and (b) a light chain variable region comprising CDR1, CDR2,and CDR3 of the respective antibodies. Other embodiments describedherein relate to antibodies that bind to the same epitope on as the VHand VL sequences described in Tables 1 and 2.

TABLE 1 HEAVY CHAIN TARGET SPECIES CDR1 CDR2 CDR3 Properdin MouseGYIFTNYPIH FIDPGGGYDEPDERFRD RGGGYYLDY (SEQ ID NO: 1) (SEQ ID NO: 2)(SEQ ID NO: 3) Properdin Mouse GFSLSTSGMGVG HIWWDDVKSYNPALKS IGDGYYSFDY(SEQ ID NO: 4) (SEQ ID NO: 5) (SEQ ID NO: 6) Properdin Mouse GYIFTTYPIHFIDPGGGYDEPDDKFRD RGDGYYFDY (SEQ ID NO: 7) (SEQ ID NO: 8) (SEQ ID NO: 9)Properdin AMGEN GDSISSGGHYWS YIYYSGSSYYNPSLKS TGDYFDY (SEQ ID NO: 10)(SEQ ID NO: 11) (SEQ ID NO: 12) Properdin AMGEN GFTFSNYGIHVIWYDGNNKYYADSVKG GGYYDSRGYYTPYYYYGMDV (SEQ ID NO: 13) (SEQ ID NO: 14)(SEQ ID NO: 15) Properdin AMGEN GFTFSCYGMH VIWYDGSNKYYADSVKG AGGATAMDV(SEQ ID NO: 16) (SEQ ID NO: 17) (SEQ ID NO: 18) Properdin AMGENGYTLTELSMH GFDPEDGETIYAQMFQG GTYYDILTGPSYYYYGLGV (SEQ ID NO: 19)(SEQ ID NO: 20) (SEQ ID NO: 21) Properdin AMGEN GGSISIYYWSYIYYSGSTNYNPSLKS WNYGDAFDI (SEQ ID NO: 22) (SEQ ID NO: 23)(SEQ ID NO: 24) Properdin Rabbit GFSFSSGYWIF GIYSGSSGTTYYANWAKGSVDGIDSYDAAFNL (SEQ ID NO: 25) (SEQ ID NO: 26) (SEQ ID NO: 27) Factor BbMouse GYTFTNYWIH YINPNTGYNDYNQKFKD GGQLGLRRAMDY (SEQ ID NO: 28)(SEQ ID NO: 29) (SEQ ID NO: 30) Factor Bb Rabbit GFDLSTYAMSAVSATTGNTYYATWAKG YASSGVGTYFDL (SEQ ID NO: 31) (SEQ ID NO: 32)(SEQ ID NO: 33) Factor Bb Rabbit GFSLSNYHLG VITYGGSTYYASWVKG RDSGGYHLDL(SEQ ID NO: 34) (SEQ ID NO: 35) (SEQ ID NO: 36) Factor Bb RabbitGFSLSSNAIN TIHTNTKTYYATWARG ADL (SEQ ID NO: 37) (SEQ ID NO: 38)(SEQ ID NO: 39) Factor C3b Mouse GYTFTSYWIN DIYPVRGITNYSEKFKNGNFGNFDAMDY (SEQ ID NO: 40) (SEQ ID NO: 41) (SEQ ID NO: 42)

TABLE 2 Light Chain TARGET SPECIES CDR1 CDR2 CDR3 Properdin MouseRASQDISFFLN YTSRYHS QHGNTLPWT (SEQ ID NO: 43) (SEQ ID NO: 44)(SEQ ID NO: 45) Properdin Mouse KASQDVSDAVA SPSYRYT QQHYSTPWT(SEQ ID NO: 46) (SEQ ID NO: 47) (SEQ ID NO: 48) Properdin MouseRSSQSLVHSNGNTYLH RSSQSLVHSNGNTYLH SQNTHVPRT (SEQ ID NO: 49)(SEQ ID NO: 50) (SEQ ID NO: 51) Properdin AMGEN RASQDISNYLA AASTLQSQKYNSAPWT (SEQ ID NO: 52) (SEQ ID NO: 53) (SEQ ID NO: 54) ProperdinAMGEN RASQGISNYLA AASTLQS QKYDSAPWT (SEQ ID NO: 55) (SEQ ID NO: 56)(SEQ ID NO: 57) Properdin Properdin Properdin Properdin RabbitQASDNIYSLLA RASTLAS QQHYDYNYLDVA (SEQ ID NO: 58) (SEQ ID NO: 59)(SEQ ID NO: 60) Factor Bb Mouse RASKSISKYLA SGSTLQS QQHDEYPWT(SEQ ID NO: 61) (SEQ ID NO: 62) (SEQ ID NO: 63) Factor Bb RabbitQASENIYSRLA YASDLAS HSYYWNSAYSDNT (SEQ ID NO: 64) (SEQ ID NO: 65)(SEQ ID NO: 66) Factor Bb Rabbit QASENIYSYLA KASYLAS LSTIASASNFDA(SEQ ID NO: 67) (SEQ ID NO: 68) (SEQ ID NO: 69) Factor Bb RabbitQSSQSVYRSNNVA EASSLAS AGGYSSSVDFFFA (SEQ ID NO: 70) (SEQ ID NO: 71)(SEQ ID NO: 72) Factor  Mouse SATSSITYIH DTSRLAS QQWSSNPPT C3b(SEQ ID NO: 73) (SEQ ID NO: 74) (SEQ ID NO: 75)

In other embodiments, an antibody described herein can comprises heavyand light chain variable regions comprising amino acid sequences thatare homologous to the amino acid sequences of the preferred antibodiesdescribed herein, and wherein the antibodies retain the desiredfunctional properties. For example, the invention provides an isolatedmonoclonal antibody, or antigen binding portion thereof, comprising aheavy chain variable region and a light chain variable region, wherein:(a) the heavy chain variable region comprises an amino acid sequencethat is at least 80% homologous to the amino acid sequence of a heavychain variable region listed in Table 1 for a respective antibody; (b)the light chain variable region comprises an amino acid sequence that isat least 80% homologous to the amino acid sequence of a light chainvariable region listed in Table 2 for the respective antibody; and (c)the antibody specifically binds to respective protein, C3b, P, Ba, orBb.

In various aspects, the antibody can be, for example, a human antibody,a humanized antibody or a chimeric antibody. In other aspects, the V_(H)and/or V_(L) amino acid sequences may be 85%, 90%, 95%, 96%, 97%, 98% or99% homologous to the sequences set forth above. An antibody havingV_(H) and V_(L) regions having high (i.e., 80% or greater) homology tothe V_(H) and V_(L) regions of the sequences set forth above, can beobtained by mutagenesis (e.g., site-directed or PCR-mediatedmutagenesis) of nucleic acid molecules encoding the amino acidsequences, followed by testing of the encoded altered antibody forretained function using the functional assays described herein.

As used herein, the percent homology between two amino acid sequences isequivalent to the percent identity between the two sequences. Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences (i.e., % homology=# ofidentical positions/total # of positions ×100), taking into account thenumber of gaps, and the length of each gap, which need to be introducedfor optimal alignment of the two sequences. The comparison of sequencesand determination of percent identity between two sequences can beaccomplished using a mathematical algorithm, as described in thenon-limiting examples below.

In certain aspects, an antibody of the invention can include a heavychain variable region comprising CDR1, CDR2 and CDR3 sequences and alight chain variable region comprising CDR1, CDR2 and CDR3 sequences,wherein one or more of these CDR sequences comprise specified amino acidsequences based on the preferred antibodies described herein, orconservative modifications thereof, and wherein the antibodies retainthe desired functional properties. Accordingly, the invention providesan isolated monoclonal antibody, or antigen binding portion thereof,comprising a heavy chain variable region comprising CDR1, CDR2, and CDR3sequences and a light chain variable region comprising CDR1, CDR2, andCDR3 sequences.

As used herein, the term “conservative sequence modifications” isintended to refer to amino acid modifications that do not significantlyaffect or alter the binding characteristics of the antibody containingthe amino acid sequence. Such conservative modifications include aminoacid substitutions, additions and deletions. Modifications can beintroduced into an antibody of the invention by standard techniquesknown in the art, such as site-directed mutagenesis and PCR-mediatedmutagenesis. Conservative amino acid substitutions are ones in which theamino acid residue is replaced with an amino acid residue having asimilar side chain. Families of amino acid residues having similar sidechains have been defined in the art. These families include amino acidswith basic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine), beta-branchedside chains (e.g., threonine, valine, isoleucine) and aromatic sidechains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, oneor more amino acid residues within the CDR regions of an antibody of theinvention can be replaced with other amino acid residues from the sameside chain family and the altered antibody can be tested for retainedfunction (i.e., the functions set forth in (c) through (j) above) usingthe functional assays described herein.

An antibody of the invention further can be prepared using an antibodyhaving one or more of the V_(H) and/or V_(L) sequences disclosed hereinas starting material to engineer a modified antibody, which modifiedantibody may have altered properties from the starting antibody. Anantibody can be engineered by modifying one or more residues within oneor both variable regions (i.e., V_(H) and/or V_(L)), for example withinone or more CDR regions and/or within one or more framework regions.Additionally or alternatively, an antibody can be engineered bymodifying residues within the constant region(s), for example to alterthe effector function(s) of the antibody.

One type of variable region engineering that can be performed is CDRgrafting. Antibodies interact with target antigens predominantly throughamino acid residues that are located in the six heavy and light chaincomplementarity determining regions (CDRs). For this reason, the aminoacid sequences within CDRs are more diverse between individualantibodies than sequences outside of CDRs. Because CDR sequences areresponsible for most antibody-antigen interactions, it is possible toexpress recombinant antibodies that mimic the properties of specificnaturally occurring antibodies by constructing expression vectors thatinclude CDR sequences from the specific naturally occurring antibodygrafted onto framework sequences from a different antibody withdifferent properties. Thus, such antibodies contain the V_(H) and V_(L)CDR sequences described in the Tables yet may contain differentframework sequences from these antibodies.

Another type of variable region modification is to mutate amino acidresidues within the V_(H) and/or V_(K) CDR1, CDR2 and/or CDR3 regions tothereby improve one or more binding properties (e.g., affinity) of theantibody of interest. Site-directed mutagenesis or PCR-mediatedmutagenesis can be performed to introduce the mutation(s) and the effecton antibody binding, or other functional property of interest, can beevaluated in in vitro or in vivo assays as described herein and providedin the Examples. Conservative modifications (as discussed above) areintroduced. The mutations may be amino acid substitutions, additions ordeletions, but are preferably substitutions. Moreover, typically no morethan one, two, three, four or five residues within a CDR region arealtered.

In general, therapeutic antibodies, once selected, can be manipulated,altered and engineered in a variety of ways for various differentreasons. For example, the inactive (non-binding) parts of an selectedantibody may be changed and manipulated in countless ways which do notat all change the defining functions of the antibody. In fact, thefunctional (protein binding part) of the antibody might be entirelysevered from the rest of the antibody. These alterations may haveutility for making the antibody easier or less costly to produce. Or,such alterations may make the antibody more chemically stable in humansubjects. These manipulations and derivations of the selected antibodiesare not new or separate inventions. Accordingly, any such manipulations,alternations and derivations of the selected genus of antibodies whichutilize the same defining characteristics of the genus itself are withinthe scope of the invention.

The invention includes compounds which constitute the functional (targetprotein binding) components of any one or several of the selected genusof antibodies, as well as the therapeutic use such compounds. Thesecompounds include, but are not limited to, whole antibodies of theselected genus, antigen-binding fragments of antibodies of the selectedgenus, and chimeric or humanized manifestations of any antibody orantibody fragment derived from the selected genus of antibodies. Suchderivations of the inventions may include, but are not limited to,truncated, linear, single-chained, an IgG fragment, a F(ab) fragment, aF(ab′) fragment, a F(ab)2 fragment, a F(ab′)2 fragment, an Fv fragmentor an scFv fragment which may be manifested from any antibody of theselected genus.

The invention includes the result of any member of the antibody genushaving its Fc region mutated at the 297 position to generate anaglycosylated antibody. The invention includes the results of anyantibody of the selected genus being engineered to elicit reducedFc-mediated effector functions. Methods of engineering may include,without limitation, amino acid mutations, amino acid additions ordeletions, glycan modification or removal, pegylation, and/ortruncation.

Methods of Administration

The invention provides methods of treatment comprising administering toa subject an effective amount of an embodiment of the invented genus ofantibodies. The subject may be an animal (a mammal such as a cow, pig,rat, or monkey) but is preferably a human. Various delivery systems areknown and can be used to administer an embodiment of the invention,(e.g., encapsulation in liposomes, microparticles, microcapsules,recombinant cells capable of expressing the compound, receptor-mediatedendocytosis, construction of a nucleic acid as part of a retroviral orother vector, etc.). Methods of introduction can be enteral orparenteral and may include, but are not limited to, intradermal,intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal,and oral routes. The compounds may be administered by any convenientroute, for example by infusion or bolus injection, by absorption throughepithelial or mucocutaneous linings (e.g., oral mucosa, rectal andintestinal mucosa, etc.) and may be administered together with otherbiologically active agents. Administration can be systemic or local.Administration can be acute or chronic (e.g., daily, weekly, monthly,etc.) or in combination with other agents.

Dosage

Administration of the invented genus of antibodies, and/or anyfunctional derivations thereof, may be by any method known in the art.Such administration may be subcutaneous, intraarticular, intramuscular,intradermal, intraperitoneal, intravenous, intranasal, or via oralroutes of administration. In one preferred embodiment, the antibody isadministered by subcutaneous injection or intravenous injection. In aspecific embodiment, the antibody is administered by subcutaneousinjection.

In one embodiment, the amount of AP antibody administered is in a dosagerange between 0.3 mg/kg to 30 mg/kg. In a more specific embodiment, theAP antibody is administered once a day in a range between 0.5 mg/kg to10 mg/kg. In another embodiment, AP antibody is administered in a dosagerange between 0.3 mg/kg to 30 mg/kg at least once a week. In yet anotherembodiment, AP antibody is administered in a dosage range between 0.3mg/kg to 30 mg/kg at least once a month. Thus, depending upon the APinhibition profile, administration regimen can be chosen.

Formulation

The compound can be administered to an individual in a formulation witha pharmaceutically acceptable excipient(s). A wide variety ofpharmaceutically acceptable excipients are known in the art and need notbe discussed in detail herein.

The compound can be incorporated into a variety of formulations fortherapeutic administration. In one example, a subject compound can beformulated into pharmaceutical compositions by combination withappropriate, pharmaceutically acceptable carriers or diluents, and canbe formulated into preparations in solid, semi-solid, liquid or gaseousforms, such as tablets, capsules, powders, granules, ointments,solutions, suppositories, injections, inhalants and aerosols.

Formulations can also be developed for subcutaneous, intraperitoneal,intravenous, and intraarticular administration.

Dosing Schedule

A compound of the present invention can be administered to an individualwith a certain frequency and for a period of time so as to achieve thedesired therapeutic effect. For example, an antibody of the presentinvention can be administered, for example, once per month, twice permonth, three times per month, every other week (qow), once per week(qw), twice per week (biw), three times per week (tiw), four times perweek, five times per week, six times per week, every other day (qod),daily (qd), twice a day (qid), or three times a day (tid), orsubstantially continuously, or continuously, over a period of timeranging from about one day to about one week, from about two weeks toabout four weeks, from about one month to about two months, from abouttwo months to about four months, from about four months to about sixmonths, or longer.

AP Specific Antibodies that Inhibit Alternative Pathway (AP)-DependentInflammation in Hemolytic Diseases

Alternative Pathway in Whole Blood—Inflammation Model

There is direct link between C3a/C5a production and activation ofneutrophils, monocytes, and platelets and release of a battery ofinflammatory cytokines, proteases, and peroxides. In this model, wholeblood from a healthy donor is subjected to AP activation via contact asa stimulus. Anaphylatoxin production, cellular activation andmeasurement of inflammatory cytokines were determined in the presenceand absence of antibodies of the current invention. Activation of cellsis related to aggregate formation and finally removal from circulationcausing cytopenia. AP antibodies of this invention are demonstrated tohave a regulatory effect on prevention of activation and cytopenia.

In order to demonstrate the effect of the activation of the alternativepathway in vivo, an ex vivo whole blood inflammation model was used.This model produces effects similar to those exhibited by the cellsinvolved in initiating and perpetuating the inflammatory response. Thewhole blood system contains the full array of complement proteins andcells responsible for carrying out the ultimate inflammatory responsewhich is the end result of alternative pathway activation. Thealternative pathway is triggered in whole blood by contact of the plasmawith the artificial surfaces of the polypropylene tubing. Even a simpleexposure of the plasma to air can trigger AP activation, and theresultant cellular activation and release of inflammatory mediators. Inthis model, blood circulation in an artificial system generatescomplement anaphylatoxins, activated cells, and inflammatory mediatorssuch as TNF-α and IL-1. Multiplex analysis further indicated theproduction of cytokines such as VEGF, IL-1, IL-17, and severalmacrophage derived cytokines. These effects in vitro can predict thedisease outcome if elevated levels of such components are found in bloodor local tissue levels.

Anti-C3b, Anti-Ba, Anti-Bb, and Anti-P antibodies have the potential todown regulate the formation of TNF-α and therefore prevent the onset andprogression of the arthritic condition. The therapeutic value of areduction of TNF-α is a known phenomenon to those of ordinary skill inthe art. However, as discussed previously, one skilled in the art cannotpredict the outcome of using any given antibody against a given proteinin the alternative pathway. Not all Anti-C3b antibodies will have atherapeutic effect. The same is true for Anti-Ba, Anti-Bb and Anti-Pantibodies unless selected using the two step process.

AP Specific Antibodies and Cellular Lysis/Inflammation in Humans

In addition to the process of developing the invented genus ofantibodies, as well as the resultant genus of antibodies, the inventionadditionally consists of a method of treating cellular lysis, cellulardamage, and inflammation in hemolytic disorders. The method comprises ofadministering to the afflicted subject a therapeutically effectiveamount of a compound which is either a member of the invented genus ofantibodies, and/or has been derived from such an antibody and utilizesthe same AP inhibiting properties as any antibody from the inventedgenus of antibodies. Such a compound, or compounds, would inhibit the APprocesses which lead to the complement activated intravascular andextravascular hemolysis. The antibodies of the claimed invention do notinhibit CP amplification loop and therefore only inhibit AP activationregardless of the target against which they have been made. Theseantibodies are claimed to inhibit only alternative pathway derived C3bbut not classical pathway derived C3b.

In other inventions, the classical pathway can use the amplification ofthe alternative pathway amplification loop and prevent C3b produced viathe classical pathway.

Intravascular Hemolysis—is caused by the deposition of C5b-9 on cellsurfaces of erythrocytes. The MAC causes cell lysis. Such a lysis ispresent on cells that are deficient in CD59. Antibodies of the claimedinvention inhibit only AP derived C5b-9 formation and not the classicalpathway derived C3b.

Extravascular Hemolysis—is caused by the deposition of C3b on the cellsurface. The C3b is responsible for effective removal of cells viaextravascular route. The antibodies of the claimed invention inhibit C3bformation and therefore inhibition of alternative pathway mediatedremoval of cells.

Paroxysmal nocturnal hemoglobinuria (“PNH”) and other hemolytic diseasesare treated using a antibody of the claimed invention which binds to orotherwise blocks the generation and/or activity of one or morecomplement components of the alternative pathway and not the classicalpathway. Such compounds include, for example, antibodies and fragmentsof the antibodies which bind to or otherwise block the generation and/oractivity of one or more complement components of the alternative pathwaybut not classical pathway, such as, for example, an antibody specific tocomplement component C3b, Properdin, Ba, and Bb. The compound is ananti-C3b antibody, an anti-properdin antibody, an anti Ba antibody, andan anti-Bb antibody. Such antibodies are further selected from the groupconsisting of Anti-C3b (murine, chimeric and humanized), an anti-Pantibody (murine, chimeric and humanized), and anti-Ba (murine, chimericand humanized), and anti-Bb (murine, chimeric, humanized) and otherfunctional fragments of such antibodies. These antibodies are requiredto have two major functions; a) ability to inhibit C3b and C5b-9formation.

It was surprising to find that a group of selected antibodies do notinhibit the amplification loop of the classical pathway and therefore donot inhibit the classical pathway in 10% NHS. These antibodies wereselected from a set of assays that specifically isolates antibodies thatare specific to the alternative pathway. These compounds inhibit thepathway upstream and therefore are a potent inhibitors of C3a, C3b, C5a,C5b, and C5b-9 formation in vitro and in ex vivo in human blood andplasma/serum. Inhibition of C3b formation by such antibodies isimportant to prevent extravascular hemolysis. Antibodies of thisinvention prevent C3b formation produced only via the AP but not CP in adose dependent fashion in human serum and whole blood. Antibodies ofthis invention also inhibit AP derived C5b-9 and sC5b-9 formation inwhole blood and/or serum. Therefore it is surprising that theseantibodies do not inhibit any amplification of the classical pathway.

The AP-inhibiting antibodies can be administered prophylactically inindividuals known to have a hemolytic disease to prevent, or helpprevent the onset of symptoms. Alternatively, the AP-inhibitingantibodies can be administered as a therapeutic regimen to an individualexperiencing symptoms of a hemolytic disease.

In another aspect, a method of increasing the proportion of damagesensitive type III red blood cells and therefore the total red bloodcell count in a patient afflicted with a hemolytic disease willincrease. The method comprises administering a compound which binds to aspecific AP protein and blocks the AP but not the CP. By increasingtotal number of erythrocytes, symptoms such as fatigue and anemia arealleviated in a patient afflicted with a hemolytic disease.

In another aspect, the present invention provides a method of renderinga subject afflicted with a hemolytic disease, transfusion-independent byadministering a compound to the subject. The, compound being selectedfrom the group consisting of anti-C3b, anti-P, anti-Ba, and anti-Bbantibodies and their functionally active antigen binding fragments whichbind the AP specific protein, compounds which block the formation of C3band C5b-9 that block the activity of one or more AP specific proteins.

It is surprising that AP specific antibodies can reduce the lysis oferythrocytes and patients are rendered transfusion-independent inaccordance with the present methods. Less C5b-9 formation is directlyrelated to less cellular damage means more cells and patients can becometransfusion independent and may not require transfusion.

In another aspect, the present invention contemplates a method ofreducing the lysis of red blood cells, the present methods reduce theamount of free hemoglobin in the blood, thereby increasing nitric oxide(NO) and prevention of kidney damage.

In another aspect, the present invention contemplates a method oftreating/preventing thrombosis in a subject by administering theantibodies of the claimed invention to prevent platelet activation,platelet lysis, removal of platelets, and formation of plateletaggregates.

In another aspect, the present invention contemplates a method oftreating pharmacological effects of preventing cell damage, wherein thecells are selected from the group comprising neutrophils, monocytes,platelets, and T-lymphocytes.

In yet another aspect, the present invention contemplates a method oftreating a subject afflicted with a hemolytic disease byadministering: 1) one or more compounds known to increase hematopoiesisin combination with 2) a compound selected from the group consisting ofcompounds that inhibit AP activation by inhibiting C3b formation andC5b-9 formation in a subject. Suitable compounds known to increasehematopoiesis include, for example, steroids, immunosuppressants (suchas, cyclosporin), anti-coagulants (such as, warfarin), folic acid, ironand the like, erythropoietin (EPO) and antithymocyte globulin (ATG) andantilymphocyte globulin (ALG). In particularly useful embodiments,erythropoietin (EPO) is administered in combination with an antibodyselected from the group consisting of anti-C3b, anti-P, anti-Ba, andanti-Bb antibodies.

In another aspect, the present disclosure provides a method of treatingone or more symptoms of hemolytic diseases in a subject where the redcells are subject to complement attack, by administering a compoundselected from the group consisting of compounds which bind to APspecific complement components, compounds which block the formation ofC3a, C5a, C5b-9 and compounds which block the activity of one or morecomplement components such as P, Ba, Bb, C3a, C5a, C5b, C6, C7, C8, andC9, said compound being administered alone or in combination with one ormore compounds known to increase hematopoiesis.

In another aspect, the methods of the present invention can selectivelyinhibit the activation of the alternative pathway in a human. TheType-AP antibody can inhibit activation of the alternative pathwaywithout affecting activation of the classical pathway or theamplification loop of the CP.

In another aspect, the alternative pathway specific antibody can beselected from the group comprising, but is not limited to, an anti-C3bantibody, and anti-Factor Ba antibody, an anti-Factor Bb antibody,anti-factor B antibody, an anti-Factor D antibody, or an anti-Properdinantibody.

In a further aspect, the alternative pathway protein that thealternative specific antibody of the present invention can bind to canbe selected from the group comprising, but is not limited to, C3b,Factor B, Factor Ba, Factor Bb, Factor D, or Properdin.

In yet another aspect, the methods of the present invention can be usedto prevent the formation of byproducts that can form as a result ofactivation of the alternative pathway in a human. In one example, themethods of the present invention can prevent the formation ofanaphylatoxins. Anaphylatoxins include, C3a and C5a. In another example,the methods of the present invention can prevent the formation of C5b-9or sC5b-9 (otherwise known as MAC). In a further example, the methods ofthe present invention can prevent the activation of neutrophils,macrophages, and platelets in a subject. In yet another example, themethods of the present invention can prevent the formation of cytokines.Cytokines can include, but are not limited to, IL-1, TNF-α, VEGF,GM-CSF.

In one aspect, the alternative pathway specific antibody of the presentinvention can be a monoclonal antibody, a polyclonal antibody, anaglycosylated antibody, or an antibody that has one or more mutations.

In another aspect, the alternative pathway specific antibody of thepresent invention can be selected from the group including, but notlimited to, human, humanized, recombinant, chimeric, de-immunized,truncated, aglycosylated, linear, single-chained, an IgG fragment, aF(ab) fragment, a F(ab′) fragment, a F(ab)2 fragment, a F(ab′)2fragment, an Fv fragment or an scFv fragment.

In another aspect, the methods of the present invention can include analternative pathway specific antibody that can have a reduced effectorfunction. Reduced effector functions can include, but are not limitedto, reduced Fc binding, lack of Fc activation, an Fc region thatcontains mutations that prevent the Fc effector functions, or theprevention of activation of platelets and cells that bear Fc receptors.

In another aspect, an effective amount of the alternative pathwayspecific antibody can be administered to the subject. In one example,the alternative pathway specific antibody or antigen binding fragmentthereof can be administered to the subject in a therapeuticallyeffective amount. In another example, the alternative pathway specificantibody or antigen binding fragment thereof can be administered to thesubject in a prophylactically effective amount. In a further example thealternative pathway specific antibody can be effective in a therapeuticsetting in vivo or ex vivo. In yet another example, the alternativepathway specific antibody can be effective in a prophylactic setting invivo or ex vivo.

In yet another aspect, the alternative pathway specific antibody of thepresent invention can contain antigen binding regions termed ascomplementarity determining regions, or CDRs. In one example, the CDRsof the alternative pathway specific antibody can be present in a fusionprotein. In another example, the CDRs of the alternative pathwayspecific antibody can be derived from a rabbit alternative pathwayspecific monoclonal antibody or a mouse alternative pathway specificmonoclonal antibody. In a further example, the CDRs of the alternativepathway specific antibody can have greater than 50% homology to thenative CDRs of the alternative pathway specific antibody.

AP Specific Antibodies and Other Diseases

The invention genus of antibodies may be used to treat any disease, ordisease condition, associated with inappropriate activation, or overactivation, of the alternative pathway. Examples of alternativecomplement pathway associated disorders are numerous. The following is alist of some, but not all, of the diseases, and/or disease symptoms andconditions, which may be ameliorated through administration of theinvention genus of antibodies.

Pathologies of the Auditory System—

Ménière's disease, in which complement factors H and B areover-expressed

Pathologies of the Cardiovascular System—

Kawasaki's disease (arteritis) Cardiac surgery complicationsHenoch-Schonlein purpura nephritis, wherein studies suggest thatgeneration of MAC may be involved in the pathogenesis of vascular injuryin a significantly large number of skin lesions and of HSP nephritis,Vascular leakage syndrome (associated with elevated c3a), Percutaneouscoronary intervention (PCI)/coronary angioplasty, Ischemia-reperfusionfollowing acute myocardial infarction, Myocardial infarction, whichelevates C3 and C4, Atherosclerosis, where C5a is present inatherosclerotic plaques, Immune complex vasculitis, in which MAC altersthe membrane integrity of endothelial cells, Arteritis, which contain C3and C4 deposits, Aneurysm, where it has been shown that C5 inhibitionattenuates injury in abdominal aortic aneurysm model, Cardiomyopathy,where c5b-9 activates TNF-α, vasculitis, where it has been shown thatC5−/− mice and factor B−/− mice do not develop disease, Takayasu'sarteritis, Dilated cardiomyopathy, where c5b-9 activates TNF-α, Venousgas embolus (VGE), Wegener's granulomatosis, Behcet's syndrome,Autoimmune cardiomyopathy, Balloon angioplasty, in which high levels ofC5a are associated with restenosis, Myocarditis, where C3a and TNF-α arepresent, Percutaneous transluminal coronary angioplasty (PTCA), IL-2induced vascular leakage syndrome, Coronary artery disease (CAD), wherethere are high C3 levels, Dressler's syndrome (postmyocardial infarctionsyndrome), in which C3d levels are elevated, Giant cell arteritis(temporal or cranial arteritis), Ischemic heart disease,Ischemia-reperfusion injury, which generates C3a and c5a,Leukocytoclastic vasculitis, in which c3d,g and Terminal complementcomplexes are present, Mesenteric artery reperfusion, where it has beenshown binding C3b attenuates injury, Microscopic polyangiitis,Pauci-immune vasculitis, associated with MAC, c3d, factor P, and factorB, Pulmonary vasculitis, Raynaud phenomenon, Post-ischemic reperfusionconditions, Pulmonary embolisms and infarcts, Restenosis following stentplacement, Subacute bacterial endocarditis, where C3d is presentVasculitis associated with rheumatoid arthritis and C3 deposits.

Pathologies of Connective Tissue—

Mixed connective tissue disease and Polymyalgia rheumatica, which C3 andC4 are deposited.

Pathologies of the Skin—

Pemphigoid, Epidermolysis bullosa acquisita, in which Factor B deficientmice display delayed and less severe blistering in a disease model,Autoimmune bullous dermatoses, Bullous pemphigoid, which is associatedwith C3 and C5, scleroderma, where C5b-9 and C5a receptors areactivated, Angioedema, Hereditary angioneurotic edema (HAE), Erythemamultiforme, Herpes gestationis, Sjogren's syndrome, with activatedc5b-9, Psoriasis, Alopecia areata, Atopic dermatitis (eczema), wherelevels of C3 and C4 are increased, Cicatricial pemphigoid, Dermatitisherpetiformis, Diffuse systemic sclerosis, Discoid lupus erythematosus,Eosinophilic spongiosis, Erythema nodosum, Lichen planus, Linear igadisease, Localized systemic sclerosis (morphea), Mucha-Habermanndisease, Occular cicatricial pemphigoid, Pemphigus, Pemphigus vulgaris,Pyoderma gangrenosum, VitiligoUrticaria.

Pathologies of the Endocrine System—

Hashimoto's thyroiditis, Diabetes mellitus type 1, in which C3, c3d, andC4 levels are increased, Stress anxiety, Pancreatitis, Addison'sdisease, Insulin resistance, which increases factor H, Diabeticangiopathy Graves' disease.

Conditions Associated with Extracorporeal Procedures—

Post-cardiopulmonary bypass inflammation, Heparin-induced extracorporealLDL precipitation (HELP), where C5a is increased, Postperfusionsyndrome, Post-operative pulmonary dysfunction, Post-pump syndrome incardiopulmonary bypass or renal bypass, which increases c5b-9, andcomplement activation during cardiopulmonary bypass operations,hemodialysis, cardiopulmonary bypass, leukopheresis, plasmapheresis,plateletpheresis, and extracorporeal membrane oxygenation (ECMO), whichcan activate SC5b-9 via alternative pathway.

Pathologies of the Gastrointestinal System—

Crohn's disease, Celiac Disease/gluten-sensitive enteropathy, associatedwith c3b, Intestinal ischemia, Inflammatory bowel disease (IBD),associated with c5a, Ulcerative colitis, where it has been shown a C5aantibody attenuates damage in colitis model, Eosinophilicgastroenteritis, Gastritis, where levels of c3b, ic3b, and C3c areincreased Pancreatitis.

Hematologic Disorders—

Catastrophic anti-phospholipid syndrome (CAPS)[96], Cold AgglutininDisease (CAD), which increases c3b, Thrombotic thrombocytopenic purpura(TTP), which increases CD46, factor H, and factor I, Idiopathicthrombocytopenic purpura, where C3 and C4 detected are on platelets,Serum sickness, where abnormal factor H leads to increased glomerular C3deposition, Endotoxemia, Sepsis, Atypical hemolytic uremic syndrome(ahus), where there is enhanced formation of c3bbb convertase andresistance to complement regulators, Paroxysmal Nocturnal Hemoglobinuria(PNH), where it has been shown a C5 antibody treatment reducedthromboembolism risk, Septic shock, sickle cell anemia, which elevatesc3b, Hypereosinophilic syndrome, which increases c5a, anti-phospholipid,Autoimmune lymphoproliferative syndrome, Dego's disease, where c5b-9 isactivated, Evan's syndrome, essential mixed cryoglobulinemia, and purered cell aplasia.

Pathologies of the Hepatic System—

Autoimmune chronic active hepatitis, which increase c3d, Infectioushepatitis, Primary biliary cirrhosis inflammation (PBC), associated withhigher c1q, C3, factor B, and properdin levels, Primary sclerosingcholangitis, where C3 is increased Autoimmune hepatitis.

Pathologies of Hypersensitivity—

Anaphylactic shock, in which blocking C3a and C5a has shown to beeffective therapy, Anaphylactoid reactions from use of radiographiccontrast media, adverse drug reaction, Allergy.

Pathologies of the Musculoskeletal System—

Osteoarthritis, Osteoporosis, Acute gouty arthritis, where C6 and MACare activated, Spondyloarthropathy, Polymyositis, Dermatomyositis, whichincreases C3b and c5b-C9, Ankylosing spondylitis, associated withincreased c3b, Arthritis, where C5a levels rise, Enthesitis-relatedarthritis, Eosinophilic fasciitis, Juvenile rheumatoid (idiopathic)arthritis, with increased c1q, C4, and MAC, Myositis, Psoriaticarthritis, where it has been shown that anti-C5a prevents arthritis,Reiter's syndrome (reactive arthritis) Relapsing polychondritis.

Pathologies of the Nervous System—

Myasthenia gravis, Multiple sclerosis (MS), Guillain Barre syndrome,which activates C3a and c5a, stroke, where C4 and sC3b-5 is elevated,Cranial nerve damage in meningitis, Variant Creutzfeldt-Jakob disease(vcjd), Neuropathic pain, Alzheimer's disease (AD), where it has beenshown that treatment with C5a receptor antagonist reduced pathology,Multifocal motor neuropathy (MMN), Huntington's disease (HD) where thereis deposition of C3 and C9 and upregulation of C5a receptors,Amyotrophic lateral sclerosis (ALS), which increases C5a and c5a,Parkinson's disease, degenerative disc disease (DDD), Idiopathicpolyneuropathy, allergic neuritis, where C3 depletion can result in lessinjury, Acute disseminated encephalomyelitis, Acute hemorrhagicleukoencephalitis, Autoimmune peripheral neuropathy, Chronicinflammatory demyelinating polyneuropathy, demyelination, wherereduction in C3 and C4 has shown to prevent demyelination, Idiopathicinflammatory demyelinating diseases, Lambert-Eaton myasthenic syndrome,Meningitis, in which C5a is correlated with prognosis and c5ar deficientmice suffered less brain damage, Miller-Fisher syndrome, Neuromyelitisoptica (NMO), Perivenous encephalomyelitis, where it has been shown C6deficient mice are unable to form MAC and exhibit no demyelination,progressive inflammatory neuropathy, opsoclonus myoclonus syndrome,Rasmussen's encephalitis, pediatric autoimmune neuropsychiatricdisorders associated with streptococcus, stiff person syndrome, Susacsyndromeanxiety.

Pathologies of Vision—

Endophthalmitis, where there is higher levels of C3a and C4a in thevitreous, Diabetic retinopathy, where there are C3d and c5b-9 depositsin choriocapillaris, Diabetic retinal microangiopathy, with C5b-9 in theretina, Histoplasmosis of the eye, Purtscher's retinopathy, Age-relatedmacular degeneration (AMD), Dry Age-Related Macular Degeneration (AMD),with elevated c3a, choroidal neurovascularization (CNV), Uveitis,Diabetic macular edema, Pathological myopia, Central retinal veinocclusion (CRVO), Retinal neovascularization, Retinal pigment epithelium(RPE), Choroidal neovascularization (CNY), Dominant drusen, where C3aand C5a promote coronial neurovasculiaztion, Photoreceptor and/orRetinal Pigmented Epithelial (RPE) loss, Stargardt's diseaseScleritis.

Oncological Pathologies—

Hemangiomas, Tumor cell metastasis.

Pathologies of the Renal System—

Glomerulonephritis, Poststreptococcal glomerulonephritis (PSGN),Goodpasture's disease, Membranous nephritis, Berger's Disease/iganephropathy, Mesangioproliferative glomerulonephritis, where c5b-9 iselevated, Membranoproliferative glomerulonephritis (Dense DepositDisease), Membranous glomerulonephritis, Renal cortical necrosis (RCN),Renal reperfusion injury, where it has been shown C5 inhibition protectsfrom renal injury, Cryoglobulinemic glomerulonephritis, ABO IncompatibleRenal Transplant, Atypical hemolytic uremic syndrome (ahus), Lupus (SLE)nephritis.

Pathologies of the Respiratory System—

Eosinophilic pneumonia, Hypersensitivity pneumonitis, Bronchiecstasis,Reactive airway disease syndrome, where it has been shown C5 and c5ardeficient mice show no airway hyperreactivity, Respiratory syncytialvirus (RSV) infection, Parainfluenza virus infection, Rhinovirusinfection, Adenovirus infection, Allergic bronchopulmonary aspergillosis(ABPA), Tuberculosis, Parasitic lung disease, Pollution-induced asthma,in which higher C3c and C4 in serum has been shown in children living inpolluted areas, Airway hyperresponsiveness (AHR), Adult respiratorydistress syndrome, which elevates C3 and c3a, Exercise-induced asthma,Cough variant asthma, occupational asthma, Allergic asthma,Pollen-induced asthma, Severe asthma, Chronic obstructive pulmonarydisease (COPD), Emphysema, bronchitis, Cystic fibrosis, Interstitiallung disease, Acute respiratory distress syndrome (ARDS),Transfusion-related acute lung injury (TRALI), Acute lung injury,Byssinosis, Asbestos-induced inflammation, Bronchoconstriction,Fibrosing alveolitis (idiopathic pulmonary fibrosis), which elevatesfactor Ba, Ischemia/reperfusion acute lung injury, Organic dustdiseases, where C3, c3d, and factor B levels increase, Pneumonia,Pathologies caused by inert dusts and minerals (e.g., silicon, coaldust, beryllium, and asbestos).

Systemic Disorders—

Systemic lupus erythematosis (SLE), Rheumatoid arthritis, AcquiredImmune Deficiency Syndrome (AIDS), Sarcoid, Systemic inflammatoryresponse syndrome (SIRS), Systemic juvenile rheumatoid arthritis, whichelevates Factor Bb and SC5b-9, Castleman's disease, Complement component2 deficiency, Multiple organ failure, Interleukin-2 induced toxicityduring IL-2 therapy, Barraquer-Simons Syndrome (acquired partiallipodystrophy).

Complications of Organ and Tissue Transplants—

Transplant rejection, where it has been shown anti-C5 antibodiesimproved graft survival, Xenograft rejection, Allotransplantation oforgans or grafts, where it has been shown C5 inhibition reduces antibodymediated rejection, Hyperacute rejection, Graft versus host disease,Hyperacute allograft rejection, Presensitized Renal Transplant—LivingDonor, Revascularization to transplants and/or replants.

Associated with Trauma—

Hemorrhagic shock, where it has been shown C5a receptor antagonistattenuates multiple organ injury, Hypovolemic shock, Spinal cord injury,Cerebral trauma and/or hemorrhage, Severe burns, where it has been shownC5a blockade improves burn-induced cardiac dysfunction, Frostbite, Crushinjury, Wound healing, Brain trauma, Cerebral ischemia reperfusion,which elevates C5 Smoke injury.

Pathologies of the Urogenital and/or Reproductive System—

Spontaneous abortion, Sensory bladder disease, Interstitial cystitis(painful bladder syndrome), Fetomaternal tolerance, Preeclampsia,Sinusitis, Complications of pregnancy, Chronic abacterial cystitis,Hemolysis, elevated liver enzymes, and low platelets (HELLP) syndrome,Infertility, Placental dysfunction and miscarriage and pre-eclampsia,Recurrent fetal loss.

Other Relevant Diseases and Conditions—

Agammaglobulinemia, antisynthetase syndrome, atopic allergy, autoimmuneenteropathy, autoimmune inner ear disease, autoimmune polyendocrinesyndrome type 1 (Whitaker's syndrome), autoimmune polyendocrine syndrometype 2 (Schmidt syndrome), autoimmune progesterone dermatitis, Balodisease/Balo concentric sclerosis, Vitelliform macular dystrophy (bestdisease), Bickerstaff's encephalitis, Blau syndrome, Cancer, chemicalinjury (due to irritant gasses and chemicals, e.g., chlorine, phosgene,sulfur dioxide, hydrogen sulfide, nitrogen dioxide, ammonia, andhydrochloric acid), chronic recurrent multifocal osteomyelitis,Churg-Strauss syndrome, Cogan syndrome, corneal neovascularization,Cushing's syndrome, cutaneous leukocytoclastic angiitis, Dercum'sdisease, fibrodysplasia ossificans progressiva, fibrogenic dustdiseases, gastrointestinal pemphigoid, Hashimoto's encephalitis,hemolytic uremic syndrome (HUS), hemoptysis, hypogammaglobulinemia,immune complex-associated inflammation, ischemia-related retinopathies,lichen sclerosus, lupoid hepatitis, juvenile lymphocytic thyroiditis,Majeed syndrome, malattia leventinese (radial drusen), neuromyotonia,North Carolina macular dystrophy, ord's thyroiditis, palindromicrheumatism, paraneoplastic cerebellar degeneration, parasitic diseases,Parry Romberg syndrome, pars planitis, Parsonage-Turner syndrome,pattern dystrophy, pernicious anaemia, POEMS syndrome, polyarteritisnodosa, proliferative nephritis, restless leg syndrome, retroperitonealfibrosis, rheumatic fever, rotational atherectomy, Schnitzler syndrome,Sorsby's fundus dystrophy, Still's disease, Surgical trauma, Sydenhamchorea, sympathetic ophthalmia, Tolosa-Hunt syndrome, transversemyelitis, undifferentiated spondyloarthropathy, vasculitis associatedwith systemic lupus erythematosus, vasculitis associated with hepatitisA, von Hippel-Lindau disease (VHL), Whipple's disease, AutoimmuneNeutropenia, Chemotherapy, Hemodialysis, Human Immunodeficiency Virus(HIV), Malaria, Epstein Barr Virus, Vitamin Deficiencies, Hypersplenism,Idiopathic Thrombocytopenic Purpura (ITP), Disseminated IntravascularCoagulation (DIC), Post-Transfusion Purpura, Neonatal Allo-ImmuneThrombocytopenia, Onyalai, Cyclic Neutropenia, Snake bites,Administration of Interferon, Administration of Tumor Necrosis Factor,administration of Radiotherapy, and application of Corticosteroids.

ITP—ITP is a relatively common hematological disorder defined by lowplatelet count, normal bone marrow and the absence of other causes ofthrombocytopenia. ITP can be diagnosed using standard clinicallaboratory tests are used, including: urinalysis, CBC with differential,hematology, coagulation, serum chemistry (includes determiningconcentration of GM-CSF and soluble GM-CSF), surfactant D, erythrocytesedimentation rate, and C-reactive protein.

Patients with chronic ITP are identified as at risk for bleeding ifplatelet the count is less than 30×10^(9/1) for those patients notreceiving corticosteroids or less than 50×10⁹/L for those patientsreceiving corticosteroids.

Antibody mediated rejection in organ transplantation, antineutrophilcytoplasmic autoantibody (ANCA) vasculitis, catastrophicantiphospholipid antibody syndrome, dense deposit disease or C3nephropathy, hemolytic cold agglutinin disease, neuromyelitis optica,nonexudative (dry) macular degeneration, Shiga toxin E. coli-relatedhemolytic uremic syndrome (STEC-HUS), systemic lupus erythematosus(SLE), thrombotic thrombocytopenic purpura (TTP)

Example 1 Cellular Assay to Demonstrate Inhibition of AP Activation in aSubject Using Exemplary Compounds of the Invention Genus of Antibodies

To assess the ability of the exemplary compounds of the presentinvention to inhibit AP activation in a in vivo-like system, anerythrocyte hemolysis assay was used. Rabbit red blood cells (rRBCs)were incubated with normal human serum (NHS) in an AP enabling buffer.The presence of rRBCs (“the foreign body”) preferentially inducesactivation of the AP, resulting in C5b-9 deposition on the erythrocytesand ultimately causing cell lysis. The extent of cell lysis is detectedbased on light scattering at optical density of 700 nm. Exemplarycompounds of the invention genus of antibodies inhibited hemolysis ofrRBCs in a dose dependent manner, as shown in FIGS. 2 and 3.

Introducing rabbit Erythrocytes (rRBC) into 10% human serum (withMg²⁺/EGTA) represent the introduction of a foreign cell surface whichinitiates the alternative complement cascade. Activation of the APresults in the formation of MAC which causes lysis of the foreign cells(the rRBCs). The selected antibodies of the present invention preventlysis of these erythrocytes. This process was quantified after examiningthe light scattering caused by intact red blood cells.

It is well established that rabbit erythrocytes specifically activatethe AP, with a resulting lysis of the rRBCs by the C5b-9 (MAC) complex.A progressive decrease in light scatter (due to lysis of intact cells)was measured at 700 nm as a function of time in a temperature-controlledELISA plate reader. The data were recorded and analyzed with aSpectraMax® 190 plate reader and SoftMax® Pro software. The results wereplotted with MicoCal® Origin Software.

As shown in FIG. 4, anti-C3b, anti-Ba, anti-Bb, and anti-P antibodies ofthe present invention inhibit AP activation and therefore lysis of rRBCin human serum only under conditions that promotes alternative pathwaydependent lysis.

Lysis of cells occurs in several diseases including hemolytic diseases.Inhibition of lysis would provide significant benefit in diseaseconditions where cell death occurs as a result of production of C5b-9(FIG. 4). Lysis of cells also is indicative of tissue injury seen inother diseases where role of complement in tissue injury appears to bewell established.

Example 2 The Antibody of the Present Invention does not Inhibit theClassical Pathway

To test the activity of the antibodies for CP inhibition,antibody-sensitized, sheep erythrocytes (sRBC) were incubated in 1%normal human serum in CP buffer (Ca²⁺/Mg²⁺). These sRBCs activate theCP, which induces lysis of cell membranes. Lysis of the cell membranesresults in a gradual decrease in light scattered by cells. When analternative pathway specific antibody of the present invention wasincubated with sRBCs at 37° C. in 1% NHS with a buffer containing Ca²⁺and Mg²⁺ (“the CP buffer”) no effect on hemolysis was observed withinthe time period beginning with the start of hemolysis and concludingwith maximal hemolysis. This implies that the alternative pathwayspecific antibody of the present invention does not affect CP hemolyticactivity in NHS (FIG. 5) and is not expected to compromise the CP'sexpected contribution to host defense against pathogens.

Monoclonal antibodies of the present invention, irrespective of thetarget antigen against which they have been raised, do not inhibit theclassical pathway. In a typical assay, antibody sensitized sheeperythrocytes are incubated with Normal Human Serum, with CP buffercontaining Ca⁺⁺. These conditions allow for selective activation of theclassical pathway. Mechanistically, the antigen-Antibody complex on thesurface of the sheep cells activates the classical complement pathwaywhich causes erythrocyte lysis.

As shown in FIG. 5, the representative antibody of the present inventionthat inhibits the AP but not the CP or the amplification loop of the CP.Development of monoclonal antibodies of this invention will leave theclassical pathway intact for host defense against infection.

Lack of inhibition of CP activation by the antibodies of the currentgenus suggests that host defense will not be compromised as classicalpathway is required for host defense. Classical pathway, uponactivation, generates C3b which is required for opsonization. In adisease state during AP activation. Thus C3b mediated opsonization isnot inhibited by the antibody of this invention.

Example 3 The Antibody of the Present Invention does not Inhibit theAmplification Process Required for the Full Potential of the ClassicalPathway

A specifically designed assay was used in order to test candidateantibodies for any inhibitory effect on any amplification process whichmay affect the full potential of the Classical Pathway. In this assay,antibody-sensitized sheep erythrocytes (sRBC) were incubated in 10%normal human serum in CP buffer (Ca²⁺/Mg²⁺) These sRBCs activate the CPvia an antibody-antigen bond complex, which induces lysis of cellmembranes. Lysis of cell membranes results in a gradual decrease inlight scattered by intact cells. When the alternative pathway specificantibody of the present invention was incubated with sRBCs at 37° C. inCa²⁺ and Mg²⁺ containing buffer (“the CP buffer”) in 10% normal humanserum (NHS), no effect on hemolysis was observed (FIG. 2 Panel B) withinthe time period beginning with the start of hemolysis and concludingwith maximal hemolysis. This implies that the alternative pathwayspecific antibody of the present invention does not affect CP hemolyticactivity in NHS and is not expected to compromise the CP's expectedcontribution to host defense against pathogens. It also implies that thealternative pathway specific antibodies of the present invention do notaffect any amplification process which may be required for the fullpotential of the CP. Accordingly, antibodies of the invention genus arenot expected to compromise the CP's full contribution to normal hostdefense to pathogens.

Monoclonal antibodies of the present invention were evaluated for theireffect on the on amplification of the alternative pathway. This was doneusing an assay of normal human serum (10% NHS with AP isolating Mg²⁺only buffer) at 37 degree C. with a fixed number of rabbit erythrocytes(Covance) in a temperature controlled ELISA plate reader capable ofreading at 700 nm. A progressive decrease in light scatter (due to lysisof intact cells) was measured at 700 nm as a function of time. The datawere recorded and analyzed with a SpectraMax® 190 plate reader andSoftMax® Pro software.

As shown in FIG. 2, panel B, the alternative pathway specific antibodyof the present invention does not inhibit amplification of the CP whichmight be initiated by the AP amplification loop. The antibody of thepresent invention does not inhibit any amplification of the CP (or theCP amplification loop, FIG. 2, panel A) and therefore is a specificinhibitor of the AP. Host defense will remain intact.

Example 4 The Antibody of the Present Invention Inhibits C3b Formationwhen AP is Activated

Alternative pathway activation generates C3b via the cleaving of C3 byAP C3 convertase. C3 is thereby split into C3b and C3a. Antibodies wereevaluated for inhibition of C3b using LPS to activate the AlternativePathway. Microtiter plates were coated with LPS (Lipopolysaccharide fromSalmonella Typhosa) 2 μg/50 μl in PBS overnight. The wells wereincubated with 1% BSA in PBS to block the unoccupied sites on the plate.Following 2 hour incubation at 37 degree, the plate was rinsed with PBSand incubated with Normal human serum (10% final concentration in APbuffer) was mixed with antibodies of the invention and incubated withLPS coated wells. The plate was again incubated for 2 hours 37° C. toallow C3b formation to occur. The plates were extensively washed withPBS, and components of the C3 convertase were detected appropriatelywith antibodies. We detected C3b with rabbit anti-human C3c at 1:2000 inblocking solution. Following incubation, the plates were rinsed with PBSand prepared with peroxidase labeled goat anti-rabbit at 1:2000 inblocking solution for C3b detection. All plates were developed with TMBfollowing extensive washing with PBS. In the presence of an AP specificantibody of the present invention inhibition of C3b formation wasobserved.

The alternative pathway specific antibodies of the present inventioninhibit formation of C3b produced in excess via the alternativecomplement pathway. C3b coated cells are generally destroyed via what isknown as extravascular hemolysis in PNH disease. Other nucleated cellscan be removed as well via the same mechanism. Thus neutropenia,leokopenia and thrombocytopenia are some examples where the end resultis the reduction in the number of cells. The genus of antibodies claimedin the current application is expected to prevent the formation of C3bresponsible for removal of cells via extravascular route. Extravascularlysis is important in indications such as paroxysmal nocturnalhemoglobinuria where C3b coated erythrocytes are removed fromcirculation via the unwanted extravascular route.

Shown in FIG. 18 is blood from PNH patient. T lymphocytes are shown inyellow, monocytes are shown in blue, and neutrophils are shown in red.These cells atin with FITC labeled CD45 to stain all leukocytes.Platelets are shown in green. As shown, all cells carry C3b suggestingthat it is CD55 may be partly absent on all cells to allow C3bdeposition. Antibodies of the current invention would inhibit C3bdeposition as shown in FIG. 6.

Example 5 The Antibody of the Present Invention Inhibits C5b-9 Formationin AP Buffer in 10% NHS

Alternative pathway activation generates C3b via the cleaving of C3 byAP C3 convertase. C3 is thereby split into C3b and C3a. AP C5 convertasecleaves C5 into C5a and C5b. The C5b molecule inserts itself into theplasma membrane and generates C5b-9 molecules on the cell surfaceleading to cellular lysis and damage of the cell wall. Antibodies wereevaluated for inhibition of C5b-9 using LPS to activate the AlternativePathway. Microtiter plates were coated with LPS (Lipopolysaccharide fromSalmonella Typhosa) 2 μg/50 μl in PBS overnight. Following 2 hourincubation at room temperature, the plate was rinsed with PBS andincubated with Normal human serum (10% final concentration in AP buffer)was mixed with antibodies of the invention and incubated with LPS coatedwells. The plate was again incubated for 2 hours 37° C. to allow C5b-9formation to occur. The plates were extensively washed with PBS, andcomponents of the C5b-9 were detected appropriately with neo antibody toC5b-9. We detected C5b-9 with mouse anti-MAC at 1:2000 in blockingsolution. All plates were developed with TMB following extensive washingwith PBS. In the presence of an AP specific antibody of the presentinvention inhibition of C5b-9 formation was observed.

As shown in FIG. 7, the alternative pathway specific antibodies of thepresent invention inhibit formation of C5b-9 produced in excess via thealternative complement pathway. C5b-9 coated cells are destroyed viaintravascular hemolysis in PNH disease. Other nucleated cells can beremoved as well via the same mechanism. Thus neutropenia, leokopenia andthrombocytopenia are some examples where the end result is the reductionin the number of cells. The genus of antibodies claimed in the currentapplication is expected to prevent the formation of C5b-9 responsiblefor removal of cells via extravascular route. Extravascular lysis isimportant in indications such as paroxysmal nocturnal hemoglobinuriawhere C5b-9 coated erythrocytes are removed from circulation via theintravascular lysis.

As shown in FIG. 10, all Blood cells were stained with CD59 and C5b-9antibodies. Cells including platelets, neutrophil, monocytes and Tlymphocytes are attacked by C5b-9. As shown for each cell type are theplots for CD59 and MAC. In this donor all cells demonstrated a similarpattern and therefore all or one cell type is sufficient to demonstratethe value of this assay for PNH detection and drug monitoring. The ratioof C5b-9 carring cells versus CD59 deficient cells appear to be similar.These cells will be attacked and would be dead. The antibody of thecurrent invention inhibit the formation of C5b-9.

Example 6 Inhibition of Formation of Inflammatory Mediators in WholeBlood Inflammation Model by Compounds (Antibodies of the Current Genus)

Alternative pathway activation generates C3b, which is cleaved from C3by AP C3 convertase. C3 is cleaved into C3b and C3a. Inhibition of C3bformation has been addressed in Example 5. Formation of C3a is measuredusing an ELISA (Quidel Corp). Antibodies of the present inventioninhibit the formation of C3a. C3a receptors, which bind C3a, are foundon monocytes. C3a is known to activate monocyte which release TNF-α, apotent inflammatory cytokine and an inflammatory mediator. TNF-α plays arole in the development and progression of arthritis. Anti-TNF-αtherapies alone have provided significant, though incomplete, benefitsfor patients with various arthritic conditions and diseases, includingrheumatoid arthritis and osteoarthritis. Inhibition of C3a formation isdirectly linked to the inhibition of monocyte activation and inhibitionof TNF-α formation and arthritis inflammation.

AP activation in whole blood replicates conditions that are primary todisease induction and progression. Blood inflammation is linked to APactivation and production of inflammatory cytokines. When whole humanblood is subjected to AP activation via an artificial trigger,inflammation in whole blood forwards to completion. This includes theformation of anaphylatoxins (e.g., C3a, C5a), the MAC complex(C5b-9/sC5b-9), activation of pro-inflammatory cells such asneutrophils, monocytes and platelets, and formation and release ofpro-inflammatory cytokines including TNF-α, IL-1β, IL-6, IL-8, andIL-17.

In this blood inflammation (BI) model, a 2 mL aliquot of freshlyisolated heparinized human blood was circulated in polyvinyl chloridetubing at 37° C. for 2 hours. Blood samples following the tubing looprotation were evaluated for C3a, C5a, and C5b-9/sC5b-9 formation.Additionally inflammation markers such as TNF-α and neutrophil elastasewere also measured.

The results shown in FIG. 8 demonstrate that antibodies of the inventioninhibit C3a formation. Elevated levels of C3a have been found in severaldiseases where significant pathology exists. Excessive C3a productionresults in excessive monocyte activation and a progressively severepathology. Many diseases where C3a is found elevated can be treated withthe antibodies of this invention. Inhibition of C3a suggests inhibitionof monocytes activation and inhibition of inflammation in vivo. Thus exvivo assays are reflective of in vivo inflammation which occurs in otherdiseases.

Results shown in FIG. 9 demonstrate that antibodies of the inventioninhibit C5a formation. C5a activates neutrophils and monocytes bybinding their respective receptors on each of these cell types.Activated neutrophils express CD11b and release elastase and areresponsible for edema in several models of inflammation. As shown inFIG. 11, the alternative pathway specific antibodies of the presentinvention inhibit neutrophil activation and, consequently, neutrophilmediated pathological outcomes in vivo. As shown in FIG. 19-25inhibition of AP activation prevents tissue inflammation, synovitis,bone and cartilage degradation.

Results from FIGS. 11, 12, and 13 show that the antibody of the currentinvention prevent cellular activation. Activation of all three majorcell types is inhibited. Aggregate formation and thrombosis is alsoinhibited by these antibodies as shown in FIG. 4.

Example 7 The Antibody of the Present Invention Inhibit Hemolysis & LDHIn Vivo

Rabbits were injected with PNH cells. The cells lysed over time andreleased hemoglobin and LDH which was measured by hemoQ and LDH wasmeasured using a kit that measures Lactate dehydrogenase was evaluated.Cytotox® LDH Kit Cat#G1781 and G1782 (Promega, Madison, Wis.) was usedfor LDH measurement. As shown in FIGS. 16 and 17, the antibody of thecurrent invention inhibits hemoglobin release and release of LDH invivo. The antibodies of the current invention inhibited erythrocytelysis and LDH formation in vivo in animals.

It is to be understood that this invention is not limited to particularembodiments described, as such may, of course, vary. It is also to beunderstood that the terminology used herein is for describing particularembodiments only, and is not intended to be limiting, since the scope ofthe present invention will be limited only by the appended claims. Allpatents, patent applications, publications listed or identified in thisdisclosure are herein incorporated by reference in their entirety.

Having described the invention, the following is claimed:
 1. A method of treating a hemolytic disorder in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an antibody that binds to properdin (P), wherein the antibody comprises: (1) a light chain variable domain that includes CDR sequences having the sequence of SEQ ID NO:49 (CDR1), SEQ ID NO:50 (CDR2), and SEQ ID NO:51 (CDR3), and a heavy chain variable domain that includes CDR sequences having the sequence of SEQ ID NO:7 (CDR1), SEQ ID NO:8 (CDR2), and SEQ ID NO:9 (CDR3), (2) a light chain variable domain that includes CDR sequences having the sequence of SEQ ID NO:52 (CDR1), SEQ ID NO:53 (CDR2), and SEQ ID NO:54 (CDR3), and a heavy chain variable domain that includes CDR sequences having the sequence of SEQ ID NO:10 (CDR1), SEQ ID NO:11 (CDR2), and SEQ ID NO:12 (CDR3), (3) a light chain variable domain that includes CDR sequences having the sequence of SEQ ID NO:55 (CDR1), SEQ ID NO:56 (CDR2), and SEQ ID NO:57 (CDR3), and a heavy chain variable domain that includes CDR sequences having the sequence of SEQ ID NO:13 (CDR1), SEQ ID NO:14 (CDR2), and SEQ ID NO:15 (CDR3), or (4) a light chain variable domain that includes CDR sequences having the sequence of SEQ ID NO:58 (CDR1), SEQ ID NO:59 (CDR2), and SEQ ID NO:60 (CDR3), and a heavy chain variable domain that includes CDR sequences having the sequence of SEQ ID NO:25 (CDR1), SEQ ID NO:26 (CDR2), and SEQ ID NO:27 (CDR3), and selectively inhibits C3a, C5a, C3b, C5b, and C5b-9 produced exclusively by the alternative pathway, without inhibiting the classical pathways ability to produce C3a, C5a, C3b, C5b, and C5b-9.
 2. The method of claim 1, the antibody being administered at an amount effective to prevent C3b formation responsible for extravascular hemolysis and C5b-9 responsible for intravascular hemolysis.
 3. The method of claim 1, the hemolytic disorder being selected from the group consisting of Paroxysmal Nocturnal Hemoglobinuria (PNH), Idiopathic Thrombocytopenic Purpura (ITP), Thrombotic Thrombocytopenic Purpura (TTP), Hemolytic-Uremic Syndrome (HUS), Disseminated Intravascular Coagulation (DIC), Antiphospholipid Syndrome (APS), Post-Transfusion Purpura, and Neonatal Allo-Immune Thrombocytopenia (NAITP).
 4. The method of claim 1, wherein the hemolytic disorder is associated with C3b induced activation of blood cells and the antibody is administered at amount effective to inhibit C3b induced activation of blood cells.
 5. The method of claim 4, wherein the activation of blood cells includes neutrophil activation, monocyte activation, platelet activation and T-lymphocyte activation.
 6. The method of claim 1, wherein the antibody is administered to the subject with one or more symptoms selected from the group consisting of: a. The subject has red blood cells opsonized with C3b; b. The subject has leukocytes opsonized with C3b; c. The subject has platelets opsonized with C3b; d. The subject has anemia; e. The subject has higher than normal levels of LDH; f. The subject has higher than normal levels of free hemoglobin; g. The subject has lower than normal levels of platelets; h. The subject has higher than normal levels of reticulocyte counts; and i. The subject has higher than normal levels of bilirubin.
 7. The method of claim 6, wherein the antibody reduces all or one of listed symptoms a-i to normal levels.
 8. The method of claim 1, wherein the subject is being treated for extravascular hemolysis.
 9. A method of treating aberrant cytopenia associated with alternative pathway activation in a subject in need thereof, the method comprising: administering to the subject a therapeutically effective amount of an antibody that binds to properdin (P), wherein the antibody comprises: (1) a light chain variable domain that includes CDR sequences having the sequence of SEQ ID NO:49 (CDR1), SEQ ID NO:50 (CDR2), and SEQ ID NO:51 (CDR3), and a heavy chain variable domain that includes CDR sequences having the sequence of SEQ ID NO:7 (CDR1), SEQ ID NO:8 (CDR2), and SEQ ID NO:9 (CDR3), (2) a light chain variable domain that includes CDR sequences having the sequence of SEQ ID NO:52 (CDR1), SEQ ID NO:53 (CDR2), and SEQ ID NO:54 (CDR3), and a heavy chain variable domain that includes CDR sequences having the sequence of SEQ ID NO:10 (CDR1), SEQ ID NO:11 (CDR2), and SEQ ID NO:12 (CDR3), (3) a light chain variable domain that includes CDR sequences having the sequence of SEQ ID NO:55 (CDR1), SEQ ID NO:56 (CDR2), and SEQ ID NO:57 (CDR3), and a heavy chain variable domain that includes CDR sequences having the sequence of SEQ ID NO:13 (CDR1), SEQ ID NO:14 (CDR2), and SEQ ID NO:15 (CDR3), or (4) a light chain variable domain that includes CDR sequences having the sequence of SEQ ID NO:58 (CDR1), SEQ ID NO:59 (CDR2), and SEQ ID NO:60 (CDR3), and a heavy chain variable domain that includes CDR sequences having the sequence of SEQ ID NO:25 (CDR1), SEQ ID NO:26 (CDR2), and SEQ ID NO:27 (CDR3), and selectively inhibits C3a, C5a, C3b, C5b, and C5b-9 produced exclusively by the alternative pathway, without inhibiting the classical pathways ability to produce C3a, C5a, C3b, C5b, and C5b-9.
 10. The method of claim 9, the aberrant cytopenia being associated with and/or resulting from a hemolytic disorder, the hemolytic disorder being selected from the group consisting of Paroxysmal Nocturnal Hemoglobinuria (PNH), Idiopathic Thrombocytopenic Purpura (ITP), Thrombotic Thrombocytopenic Purpura (TTP), Hemolytic-Uremic Syndrome (HUS), Disseminated Intravascular Coagulation (DIC), Antiphospholipid Syndrome (APS), Post-Transfusion Purpura, and Neonatal Allo-Immune Thrombocytopenia (NAITP).
 11. The method of claim 9, wherein the cytopenia includes at least one of leukocytopenia, thrombocytopenia, erythrocytopenia, leukocytopenia, lymphocytopenia, and neutropenia.
 12. A method of treating cellular and/or tissue damage caused by alternative complement pathway induced inflammation in a subject, the method comprising administering to the subject a therapeutically effective amount of an antibody that binds to properdin (P), wherein the antibody comprises: (1) a light chain variable domain that includes CDR sequences having the sequence of SEQ ID NO:49 (CDR1), SEQ ID NO:50 (CDR2), and SEQ ID NO:51 (CDR3), and a heavy chain variable domain that includes CDR sequences having the sequence of SEQ ID NO:7 (CDR1), SEQ ID NO:8 (CDR2), and SEQ ID NO:9 (CDR3), (2) a light chain variable domain that includes CDR sequences having the sequence of SEQ ID NO:52 (CDR1), SEQ ID NO:53 (CDR2), and SEQ ID NO:54 (CDR3), and a heavy chain variable domain that includes CDR sequences having the sequence of SEQ ID NO:10 (CDR1), SEQ ID NO:11 (CDR2), and SEQ ID NO:12 (CDR3), (3) a light chain variable domain that includes CDR sequences having the sequence of SEQ ID NO:55 (CDR1), SEQ ID NO:56 (CDR2), and SEQ ID NO:57 (CDR3), and a heavy chain variable domain that includes CDR sequences having the sequence of SEQ ID NO:13 (CDR1), SEQ ID NO:14 (CDR2), and SEQ ID NO:15 (CDR3), or (4) a light chain variable domain that includes CDR sequences having the sequence of SEQ ID NO:58 (CDR1), SEQ ID NO:59 (CDR2), and SEQ ID NO:60 (CDR3), and a heavy chain variable domain that includes CDR sequences having the sequence of SEQ ID NO:25 (CDR1), SEQ ID NO:26 (CDR2), and SEQ ID NO:27 (CDR3), and selectively inhibits C3a, C5a, C3b, C5b, and C5b-9 produced exclusively by the alternative pathway, without inhibiting the classical pathways ability to produce C3a, C5a, C3b, C5b, and C5b-9.
 13. A method of inhibiting lysis of neutrophils, monocytes, platelets, and/or T-lymphocytes in a subject, the method comprising administering to the subject a therapeutically effective amount of an antibody that binds to properdin (P), wherein the antibody comprises: (1) a light chain variable domain that includes CDR sequences having the sequence of SEQ ID NO:49 (CDR1), SEQ ID NO:50 (CDR2), and SEQ ID NO:51 (CDR3), and a heavy chain variable domain that includes CDR sequences having the sequence of SEQ ID NO:7 (CDR1), SEQ ID NO:8 (CDR2), and SEQ ID NO:9 (CDR3), (2) a light chain variable domain that includes CDR sequences having the sequence of SEQ ID NO:52 (CDR1), SEQ ID NO:53 (CDR2), and SEQ ID NO:54 (CDR3), and a heavy chain variable domain that includes CDR sequences having the sequence of SEQ ID NO:10 (CDR1), SEQ ID NO:11 (CDR2), and SEQ ID NO:12 (CDR3), (3) a light chain variable domain that includes CDR sequences having the sequence of SEQ ID NO:55 (CDR1), SEQ ID NO:56 (CDR2), and SEQ ID NO:57 (CDR3), and a heavy chain variable domain that includes CDR sequences having the sequence of SEQ ID NO:13 (CDR1), SEQ ID NO:14 (CDR2), and SEQ ID NO:15 (CDR3), or (4) a light chain variable domain that includes CDR sequences having the sequence of SEQ ID NO:58 (CDR1), SEQ ID NO:59 (CDR2), and SEQ ID NO:60 (CDR3), and a heavy chain variable domain that includes CDR sequences having the sequence of SEQ ID NO:25 (CDR1), SEQ ID NO:26 (CDR2), and SEQ ID NO:27 (CDR3), and selectively inhibits C3a, C5a, C3b, C5b, and C5b-9 produced exclusively by the alternative pathway, without inhibiting the classical pathways ability to produce C3a, C5a, C3b, C5b, and C5b-9 at levels sufficient to maintain normal host defense.
 14. The method of claim 13, the lysis being associated with and/or resulting from a hemolytic disorder, the hemolytic disorder being selected from the group consisting of Paroxysmal Nocturnal Hemoglobinuria (PNH), Idiopathic Thrombocytopenic Purpura (ITP), Thrombotic Thrombocytopenic Purpura (TTP), Hemolytic-Uremic Syndrome (HUS), Disseminated Intravascular Coagulation (DIC), Antiphospholipid Syndrome (APS), Post-Transfusion Purpura, and Neonatal Allo-Immune Thrombocytopenia (NAITP).
 15. A method of inhibition alternative pathway platelet activation and dysfunction in a subject in need thereof, the method comprising administering to the subject the method comprising administering to the subject a therapeutically effective amount of an antibody that binds to properdin (P), wherein the antibody comprises: (1) a light chain variable domain that includes CDR sequences having the sequence of SEQ ID NO:49 (CDR1), SEQ ID NO:50 (CDR2), and SEQ ID NO:51 (CDR3), and a heavy chain variable domain that includes CDR sequences having the sequence of SEQ ID NO:7 (CDR1), SEQ ID NO:8 (CDR2), and SEQ ID NO:9 (CDR3), (2) a light chain variable domain that includes CDR sequences having the sequence of SEQ ID NO:52 (CDR1), SEQ ID NO:53 (CDR2), and SEQ ID NO:54 (CDR3), and a heavy chain variable domain that includes CDR sequences having the sequence of SEQ ID NO:10 (CDR1), SEQ ID NO:11 (CDR2), and SEQ ID NO:12 (CDR3), (3) a light chain variable domain that includes CDR sequences having the sequence of SEQ ID NO:55 (CDR1), SEQ ID NO:56 (CDR2), and SEQ ID NO:57 (CDR3), and a heavy chain variable domain that includes CDR sequences having the sequence of SEQ ID NO:13 (CDR1), SEQ ID NO:14 (CDR2), and SEQ ID NO:15 (CDR3), or (4) a light chain variable domain that includes CDR sequences having the sequence of SEQ ID NO:58 (CDR1), SEQ ID NO:59 (CDR2), and SEQ ID NO:60 (CDR3), and a heavy chain variable domain that includes CDR sequences having the sequence of SEQ ID NO:25 (CDR1), SEQ ID NO:26 (CDR2), and SEQ ID NO:27 (CDR3), and selectively inhibits C3a, C5a, C3b, C5b, and C5b-9 produced exclusively by the alternative pathway, without inhibiting any of the classical pathways ability to produce C3a, C5a, C3b, C5b, and C5b-9.
 16. The method of claim 15, the platelet activation and dysfunction being associated with and/or resulting from a hemolytic disorder, the hemolytic disorder being selected from the group consisting of Paroxysmal Nocturnal Hemoglobinuria (PNH), Idiopathic Thrombocytopenic Purpura (ITP), Thrombotic Thrombocytopenic Purpura (TTP), Hemolytic-Uremic Syndrome (HUS), Disseminated Intravascular Coagulation (DIC), Antiphospholipid Syndrome (APS), Post-Transfusion Purpura, and Neonatal Allo-Immune Thrombocytopenia (NAITP). 